US6146887A - DNA expression vectors for use in the gene therapeutic treatment of vascular disorders - Google Patents

DNA expression vectors for use in the gene therapeutic treatment of vascular disorders Download PDF

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US6146887A
US6146887A US09/123,708 US12370898A US6146887A US 6146887 A US6146887 A US 6146887A US 12370898 A US12370898 A US 12370898A US 6146887 A US6146887 A US 6146887A
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Jurgen Schrader
Axel Godecke
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    • A—HUMAN NECESSITIES
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the invention relates to the use of DNA expression vectors for gene therapeutic: treatment of vascular disorders such as high blood pressure, arteriosclerosis, stenosis or restenosis.
  • PTCA transluminal coronary angioplasty
  • NO Nitrogen monoxide
  • NOS NO synthase
  • NO is an important messenger substance and/or signal molecule in the human body which mediates a multitude of physiological and pathophysiological effects.
  • NO is probably involved in the regulation of integrative performance, i.e. memory functions.
  • the NO formed by macrophages is capable of killing bacteria and parasites.
  • NO is formed from the endothelial cells where it carries out two important functions.
  • At least three different NOS isozymes belong to the family of NOS: the endothelial enzyme (eNOS), the brain enzyme (bNOS) and the inducible NOS (iNOS). All three isozymes are isolated by now, their primary structure, i.e. amino acid sequence, solved and the coding gene segments characterized. The essential difference between these NOS lies in their molecular weight and above all in the regulation of the expression and the enzymatic activity. Thus, eNOS and bNOS are regulated through their activity and i-NOS chiefly through expression.
  • eNOS endothelial enzyme
  • bNOS brain enzyme
  • iNOS inducible NOS
  • this enzyme has a molecular weight of 133 kDa, has a binding site for calmodulin which is dependent on free Ca ++ concentration and is present to more than 90% as membrane bound.
  • the brain enzyme is a homodimer with a molecular weight of 160 kDa per subunit which is present at less than 10% as membrane bound.
  • the calmodulin binding is dependent on free Ca ++ , i.e. both enzymes are only active when the intracellular Ca ++ concentration is increased, for example as a result of a receptor mediated Ca ++ influx.
  • iNOS the inducible NOS is also a homodimer with a molecular weight of 130 kDa per subunit.
  • the essential difference to the other isozymes is that the activity of iNOS is independent from calmodulin and therewith independent from cellular calcium. Because the turnover rate for L-arginine for iNOS is approximately 10-100 times higher than by eNOS and bNOS, INOS is also referred to as "high output" NOS. Under basal conditions, iNOS can not normally be detected; it is however strongly expressed after immunological activation through inflammation mediators and endotoxins.
  • Object of the present invention is to make an expression vector available which is suitable for gene therapeutic treatment of vascular disorders, especially high blood pressure or arteriosclerosis, stenosis or restenosis.
  • a DNA expression vector that comprises a DNA sequence which codes for a protein which expresses the biological activity of nitrogen monoxide synthase and comprises eukaryotic regulation elements, wherein the blood vessels transformed or infected with this DNA expression vector express recombinant nitrogen monoxide synthase in a therapeutically acceptable amount for the inhibition of stenosis or restenosis of said blood vessels and/or for the inhibition of the proliferation of the smooth vessel musculature.
  • the above expression vector finds use according to the invention in a method for the treatment of vascular disorders, particularly high blood pressure, arteriosclerosis or stenosis, and especially for restenosis of the coronary vessels after percutaneous, transluminal coronary angioplasty.
  • the inventors have surprisingly found th at the expression of recombinant nitrogen monoxide synthase in blood vessels which were transfected with a DNA expression vector described here leads to a therapeutically relevant inhibition of vessel stenosis and restenosis after percutaneous, transluminal coronary angioplasty.
  • the invention relates to a DNA sequence, preferably a cDNA sequence, which is obtained from mammals, preferably mice or humans, which codes for iNOS, bNOS or eNOS, preferably iNOS, activity.
  • the iNOS cDNA is preferably employed because it has a higher specific activity which is furthermore Ca independent. The enzyme activity is therefore independent from regulatory influences.
  • the DNA expression vector can comprise a sequence element which allows replication in bacteria, a sequence element which allows the replication of said vector in eukaryotic cells, preferably the SV40 replication sequence element, a polyadeylation signal and one or more introns.
  • the regulation element can comprise the promoter and/or the enhancer region from eukaryotic viruses, preferably from Cytomegalovirus or Adenovirus, and more preferably the promoter-enhancer of the Cytomegalovirus immediate early polypeptide gene.
  • the present invention relates to the treatment and prevention of vascular disorders in humans such as high blood pressure and arteriosclerosis as well as stenosis or restenosis of the blood vessels.
  • the invention particularly relates to a method for the treatment and prevention of restenosis of the coronary vessels after percutaneous, transluminal coronary angioplasty (PTCA), whereby the blood vessels are brought into contact, transfected, or infected with the DNA expression vector described above.
  • PTCA percutaneous, transluminal coronary angioplasty
  • transfection or infection of the blood vessels with a DNA expression vector can occur by every standard method known in the art and is not limited to the transfection techniques described here.
  • the invention relates to a pharmaceutical composition which comprise this DNA expression vector for the treatment or prevention of the above mentioned vascular disorders.
  • the pharmaceutical composition can comprise a pharmaceutically acceptable carrier, a stabilizing agent or buffer in addition to the DNA expression vector.
  • FIG. 1 Nucleotide and amino acid sequence of the inducible NO synthase of mouse (Mus musculus/mammalia deduced from the cloned cDNA, Genbank Accession No.: M92649);
  • FIG. 2 Nucleotide and amino acid sequence of the neuronal NO synthase of human (Homo sapiens/mammalia deduced from the cloned CDNA, Genbank Accession No.: L02881);
  • FIG. 3 Nucleotide and amino acid sequence of the endothelial NO synthase of bovine (Bos taurus/mammalia deduced from the cloned cDNA, Genbank Accession No.: M95674);
  • FIG. 4a the 2.17 Kb CMV promoter containing PvuI-PvuII fragment which was isolated from the plasmid pSP65h-CMV for the further construction of the plasmid pSCMV;
  • FIG. 4b the PvuI-PvuII fragment containing the 2.80 Kb 3' region of the ⁇ -globin gene and the SV40 origin of replication which was isolated from the plasmid pSCT GAL X-556 for the further construction of the plasmid pCMV;
  • FIG. 4c the plasmid pSCMV
  • FIG. 4d the 3.97 Kb iNOS containing HindIII-XhoI fragment according to FIG. 1;
  • FIG. 4e the plasmid pSCMV-iNOS.
  • the expression plasmid pSCMV-iNOS was cloned based on the plasmid pS065 (Promega Biotech). It contains the following functional elements:
  • the CMV promoter/enhancer was present as a DNA fragment inserted in the SmaI site of the plasmids pSP65 with blunt ends (pSP65h-CMV1).
  • the PvuI-PvuII fragment was isolated from this plasmid which carries the CMV promoter (FIG. 4a).
  • FIG. 4b A PvuI-PvuII fragment which carries the 3' region of the ⁇ -globin gene and the SV40 origin of replication (FIG. 4b) was isolated from the plasmid pSCT GAL.X-556 (see FIG. 4b). This fragment was ligated with the PvuI-PvuII fragment from pSP65h-CMV1. From the resulting plasmid (pSCMV, FIG. 4c), the following alterations were performed:
  • the vector obtained is designated as pSCMV2.
  • the iNOS cDNA was cloned as a HindIII-XhoI fragment in the XbaI-SalI digested vector pSCMV2 (FIG. 4d). Additionally, the 5' overhanging ends were converted to blunt ends by filling in after the XbaI digestion. At the 3' end, the cDNA was fused with the intron and the polyadenylation signal of the ⁇ -globin gene from chicken. The functionality of the construct was verified by transfection of SOS cells and subsequently by detection of the enzyme activity. The resulting plasmid pSCMV-iNOS (FIG. 4e) was employed for the transfection experiments. The CMV promoter imparts a constitutive expression of the iNOS gene. The activity of the enzyme made is Ca independent.
  • the established restenosis rat model of Clowes A W, Reidy M A and Clowes M M was used.
  • the common carotid artery was prepared as well as the rostral lying bifurcation at which the common carotid artery branches into the external carotid artery and internal carotid artery.
  • the external carotid artery was permanently ligated approximately 1 cm rostral from the bifurcation; the internal carotid artery was temporarily ligated by a vessel clamp.
  • the external carotid artery was opened by a cut approximately 0.5 cm rostral from the bifurcation.
  • a Fogarty 2F embolectomy catheter was lead into the common carotid artery through this opening and the endothelium of the common carotid artery was removed by three passages of the dilated catheter.
  • the denuded vessel was closed with a vessel clamp 1.5 to 2 cm caudal from the bifurcation after the removal of the catheter.
  • a polyethylene catheter was integrated in the vessel from the opening in the external carotid artery. Through this catheter 50-100 ⁇ l of a solution with DNA liposome complexes were injected within 15-20 min. into the isolated section of the common carotid artery. Care was taken that the vessel section stood under pressure from the liposome solution during the transfection.
  • the external carotid artery was permanently ligated caudal to the vessel incision, and subsequently, the blood flow over the common carotid artery and internal carotid artery was re-established by removal of the vessel clamps.

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Abstract

A DNA expression vector is described which is characterized in that it comprises a DNA sequence that codes a protein which possesses the biological activity of nitrogen monoxide synthase (NOS) and eukaryotic regulation elements, wherein said eukaryotic regulation elements result in the expression of said DNA sequence in eukaryotic cells, as well as the use of the expression vector for treatment or prevention of vascular disorders.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation application of Ser. No. 08/553,503 filed Mar. 1, 1996, now abandoned, which is the national phase application of PCT/EP95/01202 filed Mar. 31, 1995 claiming priority of German Application P44 11 402.8 filed Mar. 31, 1994. The subject matter of each of these applications is hereby incorporated by reference.
The invention relates to the use of DNA expression vectors for gene therapeutic: treatment of vascular disorders such as high blood pressure, arteriosclerosis, stenosis or restenosis.
BACKGROUND OF THE INVENTION
As percutaneous, transluminal coronary angioplasty (PTCA), one understands a method which is widely employed in the clinic in which the critical constriction of a coronary vessel (coronary stenosis) is mechanically dilated by a catheter and therewith made free again. To this day, the Achilles heal of PTCA is restenosis. This appears after weeks or months after successful PTCA in 12-43% of all treated patients (Lange, R.A. et al., Southwestern Internal Medicine Conference: Restenosis: The Achilles heal of coronary angioplasty, Am. J. Med. Sci., 265-274, 1993). A renewed PTCA or a bypass operation is the necessary consequence.
In the final analysis, the exact mechanism of restenosis is unexplained. However, of importance is the initial mechanical stretching stimulus in which, aside from destruction of the endothelial cells, tearing occurs in the arteriosclerotic wall region with simultaneous deposition of fibrin and blood platelets. The blood platelets on their part release vessel constricting and proliferation increasing metabolic products which veil the growth inhibiting factors of the smooth vessel musculature. A hyperplasty of the muscle media, i.e. restenosis, is the result.
Various strategies for the treatment of restenosis were examined in clinical studies in the past. Concerning pharmacological therapy, neither the use of anti-thrombotic agents (aspirin, heparin), antispastic agents (nifedipin, diltazem) or anliproliferative agents (colchicine) nor a lipid level sinking therapy had a favorable influence on the development of the restenosis. The development of new catheters for the minimalisation of tissue trauma (laser angioplasty, atherectomy catheter) has also brought no success in the prevention of restenosis.
There is no therapy for restenosis at the present. The reason is probably because the effective levels in blood after systemic application of a drug are too low to be able to be locally therapeutically effective.
Nitrogen monoxide (NO) is formed in mammalian cells from the amino acid L-arginine through the mediation of the enzyme NO synthase (NOS). NO is an important messenger substance and/or signal molecule in the human body which mediates a multitude of physiological and pathophysiological effects. In the central nervous system, NO is probably involved in the regulation of integrative performance, i.e. memory functions. In the gastrointestinal tract, an involvement in peristalsis is suspected. The NO formed by macrophages is capable of killing bacteria and parasites. Within the heart-circulatory system, NO is formed from the endothelial cells where it carries out two important functions. In the direction of the luminal side of the vessel it inhibits platelet aggregation and is therewith jointly responsible for the antithrombogenic properties of the vessel inner wall. On the side away from the lumen NO relaxes the smooth musculature and carries out a long-term proliferation inhibiting effect. An abolition of the endothelial NO, for example by injury of the blood vessel endothelium, leads to high blood pressure in the entire organism and is probably involved in the development of arteriosclerosis. In addition, it is important for the function of NO that its biological half-life is shorter than one second. Therewith, NO can only reach the cells in the immediate neighborhood of the location of formation, i.e. the effect of NO is locally limited.
At least three different NOS isozymes belong to the family of NOS: the endothelial enzyme (eNOS), the brain enzyme (bNOS) and the inducible NOS (iNOS). All three isozymes are isolated by now, their primary structure, i.e. amino acid sequence, solved and the coding gene segments characterized. The essential difference between these NOS lies in their molecular weight and above all in the regulation of the expression and the enzymatic activity. Thus, eNOS and bNOS are regulated through their activity and i-NOS chiefly through expression.
For the characterization of the NOS enzymes the following is illustrated:
eNOS: this enzyme has a molecular weight of 133 kDa, has a binding site for calmodulin which is dependent on free Ca++ concentration and is present to more than 90% as membrane bound.
bNOS: the brain enzyme is a homodimer with a molecular weight of 160 kDa per subunit which is present at less than 10% as membrane bound. As with the eNOS, the calmodulin binding is dependent on free Ca++, i.e. both enzymes are only active when the intracellular Ca++ concentration is increased, for example as a result of a receptor mediated Ca++ influx.
iNOS: the inducible NOS is also a homodimer with a molecular weight of 130 kDa per subunit. The essential difference to the other isozymes is that the activity of iNOS is independent from calmodulin and therewith independent from cellular calcium. Because the turnover rate for L-arginine for iNOS is approximately 10-100 times higher than by eNOS and bNOS, INOS is also referred to as "high output" NOS. Under basal conditions, iNOS can not normally be detected; it is however strongly expressed after immunological activation through inflammation mediators and endotoxins.
OBJECTS OF THE INVENTION
Object of the present invention is to make an expression vector available which is suitable for gene therapeutic treatment of vascular disorders, especially high blood pressure or arteriosclerosis, stenosis or restenosis.
SUMMARY OF THE INVENTION
The above object is solved by a DNA expression vector that comprises a DNA sequence which codes for a protein which expresses the biological activity of nitrogen monoxide synthase and comprises eukaryotic regulation elements, wherein the blood vessels transformed or infected with this DNA expression vector express recombinant nitrogen monoxide synthase in a therapeutically acceptable amount for the inhibition of stenosis or restenosis of said blood vessels and/or for the inhibition of the proliferation of the smooth vessel musculature.
The above expression vector finds use according to the invention in a method for the treatment of vascular disorders, particularly high blood pressure, arteriosclerosis or stenosis, and especially for restenosis of the coronary vessels after percutaneous, transluminal coronary angioplasty.
Furthermore, a pharmaceutical composition is made available.
The inventors have surprisingly found th at the expression of recombinant nitrogen monoxide synthase in blood vessels which were transfected with a DNA expression vector described here leads to a therapeutically relevant inhibition of vessel stenosis and restenosis after percutaneous, transluminal coronary angioplasty.
The invention relates to a DNA sequence, preferably a cDNA sequence, which is obtained from mammals, preferably mice or humans, which codes for iNOS, bNOS or eNOS, preferably iNOS, activity. The iNOS cDNA is preferably employed because it has a higher specific activity which is furthermore Ca independent. The enzyme activity is therefore independent from regulatory influences.
The DNA expression vector can comprise a sequence element which allows replication in bacteria, a sequence element which allows the replication of said vector in eukaryotic cells, preferably the SV40 replication sequence element, a polyadeylation signal and one or more introns.
The regulation element can comprise the promoter and/or the enhancer region from eukaryotic viruses, preferably from Cytomegalovirus or Adenovirus, and more preferably the promoter-enhancer of the Cytomegalovirus immediate early polypeptide gene.
Additionally, the present invention relates to the treatment and prevention of vascular disorders in humans such as high blood pressure and arteriosclerosis as well as stenosis or restenosis of the blood vessels. The invention particularly relates to a method for the treatment and prevention of restenosis of the coronary vessels after percutaneous, transluminal coronary angioplasty (PTCA), whereby the blood vessels are brought into contact, transfected, or infected with the DNA expression vector described above.
The transfection or infection of the blood vessels with a DNA expression vector can occur by every standard method known in the art and is not limited to the transfection techniques described here.
Furthermore, the invention relates to a pharmaceutical composition which comprise this DNA expression vector for the treatment or prevention of the above mentioned vascular disorders. The pharmaceutical composition can comprise a pharmaceutically acceptable carrier, a stabilizing agent or buffer in addition to the DNA expression vector. The following Figures serve to illustrate the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Nucleotide and amino acid sequence of the inducible NO synthase of mouse (Mus musculus/mammalia deduced from the cloned cDNA, Genbank Accession No.: M92649);
FIG. 2 Nucleotide and amino acid sequence of the neuronal NO synthase of human (Homo sapiens/mammalia deduced from the cloned CDNA, Genbank Accession No.: L02881);
FIG. 3 Nucleotide and amino acid sequence of the endothelial NO synthase of bovine (Bos taurus/mammalia deduced from the cloned cDNA, Genbank Accession No.: M95674);
FIG. 4a the 2.17 Kb CMV promoter containing PvuI-PvuII fragment which was isolated from the plasmid pSP65h-CMV for the further construction of the plasmid pSCMV;
FIG. 4b the PvuI-PvuII fragment containing the 2.80 Kb 3' region of the β-globin gene and the SV40 origin of replication which was isolated from the plasmid pSCT GAL X-556 for the further construction of the plasmid pCMV;
FIG. 4c the plasmid pSCMV;
FIG. 4d the 3.97 Kb iNOS containing HindIII-XhoI fragment according to FIG. 1;
FIG. 4e the plasmid pSCMV-iNOS.
DETAILED DESCRIPTION OF THE INVENTION
The following Examples should more closely illustrate the invention without limiting it to them.
EXAMPLE 1
I. Elements of an Expression Vector
The expression plasmid pSCMV-iNOS was cloned based on the plasmid pS065 (Promega Biotech). It contains the following functional elements:
promoter/enhancer of the human Cytomegalovirus immediate early polypeptide (Pos. 216-809/ Genbank Accession No.: K03104)
cDNA of the inducible NO synthase of the mouse (Pos. 127-4110/ Genbank Accession No.: M92649)
genomic sequences of β-globin gene of rabbit (Pos. 905-2080) which contains intron 2, exon 3 and the polyadenylation signal (Pos. 905-1827 are stored under Genbank Accession No.: J00659)
the origin of replication of SV40 virus (Pos. 130 [SV40 early map] or Pos. 5176-130 of the circular SV40 map Genbank Accession No.: V01380).
II. Production of an Expression Vector
The CMV promoter/enhancer was present as a DNA fragment inserted in the SmaI site of the plasmids pSP65 with blunt ends (pSP65h-CMV1). The PvuI-PvuII fragment was isolated from this plasmid which carries the CMV promoter (FIG. 4a).
A PvuI-PvuII fragment which carries the 3' region of the β-globin gene and the SV40 origin of replication (FIG. 4b) was isolated from the plasmid pSCT GAL.X-556 (see FIG. 4b). This fragment was ligated with the PvuI-PvuII fragment from pSP65h-CMV1. From the resulting plasmid (pSCMV, FIG. 4c), the following alterations were performed:
deletion of the vector sequences between HindIII and PvuII with simultaneous insertion of a ClaI linker. Thereby, the starting cleavage sites PvuII and HindIII were lost.
conversion of the XhoI cleavage site into a PvuI cleavage site.
The vector obtained is designated as pSCMV2.
The iNOS cDNA was cloned as a HindIII-XhoI fragment in the XbaI-SalI digested vector pSCMV2 (FIG. 4d). Additionally, the 5' overhanging ends were converted to blunt ends by filling in after the XbaI digestion. At the 3' end, the cDNA was fused with the intron and the polyadenylation signal of the β-globin gene from chicken. The functionality of the construct was verified by transfection of SOS cells and subsequently by detection of the enzyme activity. The resulting plasmid pSCMV-iNOS (FIG. 4e) was employed for the transfection experiments. The CMV promoter imparts a constitutive expression of the iNOS gene. The activity of the enzyme made is Ca independent.
III. Production of DNA Liposome Complexes
The cultivation of Sendi virus occurred in the chorionallantis fluid of fertilized chicken eggs (d10-d12 of the embryonic development) according to standard methods (Nakanishi M., Uchida T., Sugawa H., Ishiura M., Okada Y., Exp. Cell Res. 159, 399-409).
Phosphatidylcholine, phosphatidylserine and cholesterol were dissolved in tetrahydrofuran, mixed in a molar ratio of 4.8:1:2 and evaporated under nitrogen. 10 mg of the dried lipids were suspended by vortexing in 200 μl pSCMV-iNOS [dissolved in BSS (140 mM NaCl, 5.4 mM KCl and 10 mM Tris-HCl, pH 7.69)] (conc. 1 μg/μl). The liposomes obtained were incubated with 4 ml Sendi virus (Z strain) (16000 hemagglutination units (HAU)/ml) for 3 h at 37° C. under light shaking and subsequently sonicated for 20" in the ultrasonic bath. The viruses were inactivated before use with LTV light (11 J/m2 ×s). The suspension obtained was loaded on two saccharose gradients (1 ml 60%, 1 ml 40%, 8 ml 30% saccharose in BSS) and centrifuged for 3 h at 30,000 rpm in the SW40 rotor (Beckman Instruments) at 4° C. The top 2 ml were collected and employed for the transfection experiments.
IV. Transfection Protocol
To investigate the effects of a local over-expression of iNOS on the proliferation of the smooth vessel musculature after endothelial injury in vivo, the established restenosis rat model of Clowes A W, Reidy M A and Clowes M M (Lab. Invest. 49, 327-333, 1983) was used. In the neck region, the common carotid artery was prepared as well as the rostral lying bifurcation at which the common carotid artery branches into the external carotid artery and internal carotid artery. The external carotid artery was permanently ligated approximately 1 cm rostral from the bifurcation; the internal carotid artery was temporarily ligated by a vessel clamp. The external carotid artery was opened by a cut approximately 0.5 cm rostral from the bifurcation. A Fogarty 2F embolectomy catheter was lead into the common carotid artery through this opening and the endothelium of the common carotid artery was removed by three passages of the dilated catheter.
For the "therapy group", the following method was used. The denuded vessel was closed with a vessel clamp 1.5 to 2 cm caudal from the bifurcation after the removal of the catheter. A polyethylene catheter was integrated in the vessel from the opening in the external carotid artery. Through this catheter 50-100 μl of a solution with DNA liposome complexes were injected within 15-20 min. into the isolated section of the common carotid artery. Care was taken that the vessel section stood under pressure from the liposome solution during the transfection. After removal of the transfection catheter, the external carotid artery was permanently ligated caudal to the vessel incision, and subsequently, the blood flow over the common carotid artery and internal carotid artery was re-established by removal of the vessel clamps.
V. Morphological Analysis of the Vessels
At various time points after the transfection of the common carotid artery (to 21d), rats were examined with respect to the morphological changes in the vessels. The vessels were fixed by per-fusion with 3% paraformaldehyde in PBS (140 mM NaCl, 10 mM Na phosphate, pH 7.5) and sectioned. The degree of the proliferation was compared with control treated vessels. A reduction of the stenosis formation by up to 60% in the treated animals was demonstrated. This study shows for the first time that in this animal model the local over-expression of a gene results in a therapeutically relevant inhibition of vessel stenosis and demonstrates the special roll of NO for the inhibition of the proliferation of smooth vessel musculature.
__________________________________________________________________________
-                   - #             SEQUENCE LISTING                      
  - -  - - <160> NUMBER OF SEQ ID NOS: 6                                  
  - - <210> SEQ ID NO 1                                                   
 <211> LENGTH: 4110                                                       
 <212> TYPE: DNA                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 1                                                   
  - - gagactctgg ccccacggga cacagtgtca ctggtttgaa acttctcagc ca -         
#ccttggtg     60                                                          
   - - aagggactga gctgttagag acacttctga ggctcctcac gcttgggtct tg -        
#ttcactcc    120                                                          
   - - acggagtagc ctagtcaact gcaagagaac ggagaacgtt ggatttggag ca -        
#gaagtgca    180                                                          
   - - aagtctcaga catggcttgc ccctggaagt ttctcttcaa agtcaaatcc ta -        
#ccaaagtg    240                                                          
   - - acctgaaaga ggaaaaggac attaacaaca acgtgaagaa aaccccttgt gc -        
#tgttctca    300                                                          
   - - gcccaacaat acaagatgac cctaagagtc accaaaatgg ctccccgcag ct -        
#cctcactg    360                                                          
   - - ggacagcaca gaatgttcca gaatccctgg acaagctgca tgtgacatcg ac -        
#ccgtccac    420                                                          
   - - agtatgtgag gatcaaaaac tggggcagtg gagagatttt gcatgacact ct -        
#tcaccaca    480                                                          
   - - aggccacatc ggatttcact tgcaagtcca agtcttgctt ggggtccatc at -        
#gaacccca    540                                                          
   - - agagtttgac cagaggaccc agagacaagc ctacccctct ggaggagctc ct -        
#gcctcatg    600                                                          
   - - ccattgagtt catcaaccag tattatggct cctttaaaga ggcaaaaata ga -        
#ggaacatc    660                                                          
   - - tggccaggct ggaagctgta acaaaggaaa tagaaacaac aggaacctac ca -        
#gctcactc    720                                                          
   - - tggatgagct catctttgcc accaagatgg cctggaggaa tgcccctcgc tg -        
#catcggca    780                                                          
   - - ggatccagtg gtccaacctg caggtctttg acgctcggaa ctgtagcaca gc -        
#acaggaaa    840                                                          
   - - tgtttcagca catctgcaga cacatacttt atgccaccaa caatggcaac at -        
#caggtcgg    900                                                          
   - - ccatcactgt gttcccccag cggagtgacg gcaaacatga cttcaggctc tg -        
#gaattcac    960                                                          
   - - agctcatccg gtacgctggc taccagatgc ccgatggcac catcagaggg ga -        
#tgctgcca   1020                                                          
   - - ccttggagtt cacccagttg tgcatcgacc taggctggaa gccccgctat gg -        
#ccgctttg   1080                                                          
   - - atgtgctgcc tctggtcttg caagctgatg gtcaagatcc agaggtcttt ga -        
#aatccctc   1140                                                          
   - - ctgatcttgt gttggaggtg accatggagc atcccaagta cgagtggttc ca -        
#ggagctcg   1200                                                          
   - - ggttgaagtg gtatgcactg cctgccgtgg ccaacatgct actggaggtg gg -        
#tggcctcg   1260                                                          
   - - aattcccagc ctgccccttc aatggttggt acatgggcac cgagattgga gt -        
#tcgagact   1320                                                          
   - - tctgtgacac acagcgctac aacatcctgg aggaagtggg ccgaaggatg gg -        
#cctggaga   1380                                                          
   - - cccacacact ggcctccctc tggaaagacc gggctgtcac ggagatcaat gt -        
#ggctgtgc   1440                                                          
   - - tccatagttt ccagaagcag aatgtgacca tcatggacca ccacacagcc tc -        
#agagtcct   1500                                                          
   - - tcatgaagca catgcagaat gagtaccggg cccgtggagg ctgcccggca ga -        
#ctggattt   1560                                                          
   - - ggctggtccc tccagtgtct gggagcatca cccctgtgtt ccaccaggag at -        
#gttgaact   1620                                                          
   - - atgtcctatc tccattctac tactaccaga tcgagccctg gaagacccac at -        
#ctggcaga   1680                                                          
   - - atgagaagct gaggcccagg aggagagaga tccgatttag agtcttggtg aa -        
#agtggtgt   1740                                                          
   - - tctttgcttc catgctaatg cgaaaggtca tggcttcacg ggtcagagcc ac -        
#agtcctct   1800                                                          
   - - ttgctactga gacagggaag tctgaagcac tagccaggga cctggccacc tt -        
#gttcagct   1860                                                          
   - - acgccttcaa caccaaggtt gtctgcatgg accagtataa ggcaagcacc tt -        
#ggaagagg   1920                                                          
   - - agcaactact gctggtggtg acaagcacat ttgggaatgg agactgtccc ag -        
#caatgggc   1980                                                          
   - - agactctgaa gaaatctctg ttcatgctta gagaactcaa ccacaccttc ag -        
#gtatgctg   2040                                                          
   - - tgtttggcct tggctccagc atgtaccctc agttctgcgc ctttgctcat ga -        
#catcgacc   2100                                                          
   - - agaagctgtc ccacctggga gcctctcagc ttgccccaac aggagaaggg ga -        
#cgaactca   2160                                                          
   - - gtgggcagga ggatgccttc cgcagctggg ctgtacaaac cttccgggca gc -        
#ctgtgaga   2220                                                          
   - - cctttgatgt ccgaagcaaa catcacattc agatcccgaa acgcttcact tc -        
#caatgcaa   2280                                                          
   - - catgggagcc acagcaatat aggctcatcc agagcccgga gcctttagac ct -        
#caacagag   2340                                                          
   - - ccctcagcag catccatgca aagaacgtgt ttaccatgag gctgaaatcc ca -        
#gcagaatc   2400                                                          
   - - tgcagagtga aaagtccagc cgcaccaccc tcctcgttca gctcaccttc ga -        
#gggcagcc   2460                                                          
   - - gagggcccag ctacctgcct ggggaacacc tggggatctt cccaggcaac ca -        
#gaccgccc   2520                                                          
   - - tggtgcaggg aatcttggag cgagttgtgg attgtcctac accacaccaa ac -        
#tgtgtgcc   2580                                                          
   - - tggaggttct ggatgagagc ggcagctact gggtcaaaga caagaggctg cc -        
#cccctgct   2640                                                          
   - - cactcagcca agccctcacc tacttcctgg acattacgac ccctcccacc ca -        
#gctgcagc   2700                                                          
   - - tccacaagct ggctcgcttt gccacggacg agacggatag gcagagattg ga -        
#ggccttgt   2760                                                          
   - - gtcagccctc agagtacaat gactggaagt tcagcaacaa ccccacgttc ct -        
#ggaggtgc   2820                                                          
   - - ttgaagagtt cccttccttg catgtgcccg ctgccttcct gctgtcgcag ct -        
#ccctatct   2880                                                          
   - - tgaagccccg ctactactcc atcagctcct cccaggacca caccccctcg ga -        
#ggttcacc   2940                                                          
   - - tcactgtggc cgtggtcacc taccgcaccc gagatggtca gggtcccctg ca -        
#ccatggtg   3000                                                          
   - - tctgcagcac ttggatcagg aacctgaagc cccaggaccc agtgccctgc tt -        
#tgtgcgaa   3060                                                          
   - - gtgtcagtgg cttccagctc cctgaggacc cctcccagcc ttgcatcctc at -        
#tgggcctg   3120                                                          
   - - gtacgggcat tgctcccttc cgaagtttct ggcagcagcg gctccatgac tc -        
#ccagcaca   3180                                                          
   - - aagggctcaa aggaggccgc atgagcttgg tgtttgggtg ccggcacccg ga -        
#ggaggacc   3240                                                          
   - - acctctatca ggaagaaatg caggagatgg tccgcaagag agtgctgttc ca -        
#ggtgcaca   3300                                                          
   - - caggctactc ccggctgccc ggcaaaccca aggtctacgt tcaggacatc gt -        
#gcaaaagc   3360                                                          
   - - agctggccaa tgaggtactc agcgtgctcc acggggagca gggccacctc ta -        
#catttgcg   3420                                                          
   - - gagatgtgcg catggctcgg gatgtggcta ccacattgaa gaagctggtg gc -        
#caccaagc   3480                                                          
   - - tgaacttgag cgaggagcag gtggaagact atttcttcca gctcaagagc ca -        
#gaaacgtt   3540                                                          
   - - atcatgaaga tatcttcggt gcagtctttt cctatggggc aaaaaagggc ag -        
#cgccttgg   3600                                                          
   - - aggagcccaa agccacgagg ctctgacagc ccagagttcc agcttctggc ac -        
#tgagtaaa   3660                                                          
   - - gataatggtg aggggcttgg ggagacagcg aaatgcaatc ccccccaagc cc -        
#ctcatgtc   3720                                                          
   - - attcccccct cctccaccct accaagtagt attgtattat tgtggactac ta -        
#aatctctc   3780                                                          
   - - tcctctcctc cctcccctct ctccctttcc tcccttcttc tccactcccc ag -        
#ctccctcc   3840                                                          
   - - ttctccttct cctcctttgc ctctcactct tccttggagc tgagagcaga ga -        
#aaaactca   3900                                                          
   - - acctcctgac tgaagcactt tgggtcacca ccaggaggca ccatgccgcc gc -        
#tctaatac   3960                                                          
   - - ttagctgcac tatgtacaga tatttatact tcatatttaa gaaaacagat ac -        
#ttttgtct   4020                                                          
   - - actcccaatg atggcttggg cctttcctgt ataattcctt gatgaaaaat at -        
#ttatataa   4080                                                          
   - - aatacatttt attttaatca aaaaaaaaaa         - #                  - #  
       4110                                                               
  - -  - - <210> SEQ ID NO 2                                              
 <211> LENGTH: 1144                                                       
 <212> TYPE: PRT                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 2                                                   
  - - Met Ala Cys Pro Trp Lys Phe Leu Phe Lys Va - #l Lys Ser Tyr Gln Ser 
   1               5 - #                 10 - #                 15        
  - - Asp Leu Lys Glu Glu Lys Asp Ile Asn Asn As - #n Val Lys Lys Thr Pro 
              20     - #             25     - #             30            
  - - Cys Ala Val Leu Ser Pro Thr Ile Gln Asp As - #p Pro Lys Ser His Gln 
          35         - #         40         - #         45                
  - - Asn Gly Ser Pro Gln Leu Leu Thr Gly Thr Al - #a Gln Asn Val Pro Glu 
      50             - #     55             - #     60                    
  - - Ser Leu Asp Lys Leu His Val Thr Ser Thr Ar - #g Pro Gln Tyr Val Arg 
  65                 - # 70                 - # 75                 - # 80 
  - - Ile Lys Asn Trp Gly Ser Gly Glu Ile Leu Hi - #s Asp Thr Leu His His 
                  85 - #                 90 - #                 95        
  - - Lys Ala Thr Ser Asp Phe Thr Cys Lys Ser Ly - #s Ser Cys Leu Gly Ser 
             100      - #           105      - #           110            
  - - Ile Met Asn Pro Lys Ser Leu Thr Arg Gly Pr - #o Arg Asp Lys Pro Thr 
         115          - #       120          - #       125                
  - - Pro Leu Glu Glu Leu Leu Pro His Ala Ile Gl - #u Phe Ile Asn Gln Tyr 
     130              - #   135              - #   140                    
  - - Tyr Gly Ser Phe Lys Glu Ala Lys Ile Glu Gl - #u His Leu Ala Arg Leu 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - Glu Ala Val Thr Lys Glu Ile Glu Thr Thr Gl - #y Thr Tyr Gln Leu    
Thr                                                                       
                  165  - #               170  - #               175       
  - - Leu Asp Glu Leu Ile Phe Ala Thr Lys Met Al - #a Trp Arg Asn Ala Pro 
             180      - #           185      - #           190            
  - - Arg Cys Ile Gly Arg Ile Gln Trp Ser Asn Le - #u Gln Val Phe Asp Ala 
         195          - #       200          - #       205                
  - - Arg Asn Cys Ser Thr Ala Gln Glu Met Phe Gl - #n His Ile Cys Arg His 
     210              - #   215              - #   220                    
  - - Ile Leu Tyr Ala Thr Asn Asn Gly Asn Ile Ar - #g Ser Ala Ile Thr Val 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Phe Pro Gln Arg Ser Asp Gly Lys His Asp Ph - #e Arg Leu Trp Asn    
Ser                                                                       
                  245  - #               250  - #               255       
  - - Gln Leu Ile Arg Tyr Ala Gly Tyr Gln Met Pr - #o Asp Gly Thr Ile Arg 
             260      - #           265      - #           270            
  - - Gly Asp Ala Ala Thr Leu Glu Phe Thr Gln Le - #u Cys Ile Asp Leu Gly 
         275          - #       280          - #       285                
  - - Trp Lys Pro Arg Tyr Gly Arg Phe Asp Val Le - #u Pro Leu Val Leu Gln 
     290              - #   295              - #   300                    
  - - Ala Asp Gly Gln Asp Pro Glu Val Phe Glu Il - #e Pro Pro Asp Leu Val 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - Leu Glu Val Thr Met Glu His Pro Lys Tyr Gl - #u Trp Phe Gln Glu    
Leu                                                                       
                  325  - #               330  - #               335       
  - - Gly Leu Lys Trp Tyr Ala Leu Pro Ala Val Al - #a Asn Met Leu Leu Glu 
             340      - #           345      - #           350            
  - - Val Gly Gly Leu Glu Phe Pro Ala Cys Pro Ph - #e Asn Gly Trp Tyr Met 
         355          - #       360          - #       365                
  - - Gly Thr Glu Ile Gly Val Arg Asp Phe Cys As - #p Thr Gln Arg Tyr Asn 
     370              - #   375              - #   380                    
  - - Ile Leu Glu Glu Val Gly Arg Arg Met Gly Le - #u Glu Thr His Thr Leu 
 385                 3 - #90                 3 - #95                 4 -  
#00                                                                       
   - - Ala Ser Leu Trp Lys Asp Arg Ala Val Thr Gl - #u Ile Asn Val Ala    
Val                                                                       
                  405  - #               410  - #               415       
  - - Leu His Ser Phe Gln Lys Gln Asn Val Thr Il - #e Met Asp His His Thr 
             420      - #           425      - #           430            
  - - Ala Ser Glu Ser Phe Met Lys His Met Gln As - #n Glu Tyr Arg Ala Arg 
         435          - #       440          - #       445                
  - - Gly Gly Cys Pro Ala Asp Trp Ile Trp Leu Va - #l Pro Pro Val Ser Gly 
     450              - #   455              - #   460                    
  - - Ser Ile Thr Pro Val Phe His Gln Glu Met Le - #u Asn Tyr Val Leu Ser 
 465                 4 - #70                 4 - #75                 4 -  
#80                                                                       
   - - Pro Phe Tyr Tyr Tyr Gln Ile Glu Pro Trp Ly - #s Thr His Ile Trp    
Gln                                                                       
                  485  - #               490  - #               495       
  - - Asn Glu Lys Leu Arg Pro Arg Arg Arg Glu Il - #e Arg Phe Arg Val Leu 
             500      - #           505      - #           510            
  - - Val Lys Val Val Phe Phe Ala Ser Met Leu Me - #t Arg Lys Val Met Ala 
         515          - #       520          - #       525                
  - - Ser Arg Val Arg Ala Thr Val Leu Phe Ala Th - #r Glu Thr Gly Lys Ser 
     530              - #   535              - #   540                    
  - - Glu Ala Leu Ala Arg Asp Leu Ala Thr Leu Ph - #e Ser Tyr Ala Phe Asn 
 545                 5 - #50                 5 - #55                 5 -  
#60                                                                       
   - - Thr Lys Val Val Cys Met Asp Gln Tyr Lys Al - #a Ser Thr Leu Glu    
Glu                                                                       
                  565  - #               570  - #               575       
  - - Glu Gln Leu Leu Leu Val Val Thr Ser Thr Ph - #e Gly Asn Gly Asp Cys 
             580      - #           585      - #           590            
  - - Pro Ser Asn Gly Gln Thr Leu Lys Lys Ser Le - #u Phe Met Leu Arg Glu 
         595          - #       600          - #       605                
  - - Leu Asn His Thr Phe Arg Tyr Ala Val Phe Gl - #y Leu Gly Ser Ser Met 
     610              - #   615              - #   620                    
  - - Tyr Pro Gln Phe Cys Ala Phe Ala His Asp Il - #e Asp Gln Lys Leu Ser 
 625                 6 - #30                 6 - #35                 6 -  
#40                                                                       
   - - His Leu Gly Ala Ser Gln Leu Ala Pro Thr Gl - #y Glu Gly Asp Glu    
Leu                                                                       
                  645  - #               650  - #               655       
  - - Ser Gly Gln Glu Asp Ala Phe Arg Ser Trp Al - #a Val Gln Thr Phe Arg 
             660      - #           665      - #           670            
  - - Ala Ala Cys Glu Thr Phe Asp Val Arg Ser Ly - #s His His Ile Gln Ile 
         675          - #       680          - #       685                
  - - Pro Lys Arg Phe Thr Ser Asn Ala Thr Trp Gl - #u Pro Gln Gln Tyr Arg 
     690              - #   695              - #   700                    
  - - Leu Ile Gln Ser Pro Glu Pro Leu Asp Leu As - #n Arg Ala Leu Ser Ser 
 705                 7 - #10                 7 - #15                 7 -  
#20                                                                       
   - - Ile His Ala Lys Asn Val Phe Thr Met Arg Le - #u Lys Ser Gln Gln    
Asn                                                                       
                  725  - #               730  - #               735       
  - - Leu Gln Ser Glu Lys Ser Ser Arg Thr Thr Le - #u Leu Val Gln Leu Thr 
             740      - #           745      - #           750            
  - - Phe Glu Gly Ser Arg Gly Pro Ser Tyr Leu Pr - #o Gly Glu His Leu Gly 
         755          - #       760          - #       765                
  - - Ile Phe Pro Gly Asn Gln Thr Ala Leu Val Gl - #n Gly Ile Leu Glu Arg 
     770              - #   775              - #   780                    
  - - Val Val Asp Cys Pro Thr Pro His Gln Thr Va - #l Cys Leu Glu Val Leu 
 785                 7 - #90                 7 - #95                 8 -  
#00                                                                       
   - - Asp Glu Ser Gly Ser Tyr Trp Val Lys Asp Ly - #s Arg Leu Pro Pro    
Cys                                                                       
                  805  - #               810  - #               815       
  - - Ser Leu Ser Gln Ala Leu Thr Tyr Phe Leu As - #p Ile Thr Thr Pro Pro 
             820      - #           825      - #           830            
  - - Thr Gln Leu Gln Leu His Lys Leu Ala Arg Ph - #e Ala Thr Asp Glu Thr 
         835          - #       840          - #       845                
  - - Asp Arg Gln Arg Leu Glu Ala Leu Cys Gln Pr - #o Ser Glu Tyr Asn Asp 
     850              - #   855              - #   860                    
  - - Trp Lys Phe Ser Asn Asn Pro Thr Phe Leu Gl - #u Val Leu Glu Glu Phe 
 865                 8 - #70                 8 - #75                 8 -  
#80                                                                       
   - - Pro Ser Leu His Val Pro Ala Ala Phe Leu Le - #u Ser Gln Leu Pro    
Ile                                                                       
                  885  - #               890  - #               895       
  - - Leu Lys Pro Arg Tyr Tyr Ser Ile Ser Ser Se - #r Gln Asp His Thr Pro 
             900      - #           905      - #           910            
  - - Ser Glu Val His Leu Thr Val Ala Val Val Th - #r Tyr Arg Thr Arg Asp 
         915          - #       920          - #       925                
  - - Gly Gln Gly Pro Leu His His Gly Val Cys Se - #r Thr Trp Ile Arg Asn 
     930              - #   935              - #   940                    
  - - Leu Lys Pro Gln Asp Pro Val Pro Cys Phe Va - #l Arg Ser Val Ser Gly 
 945                 9 - #50                 9 - #55                 9 -  
#60                                                                       
   - - Phe Gln Leu Pro Glu Asp Pro Ser Gln Pro Cy - #s Ile Leu Ile Gly    
Pro                                                                       
                  965  - #               970  - #               975       
  - - Gly Thr Gly Ile Ala Pro Phe Arg Ser Phe Tr - #p Gln Gln Arg Leu His 
             980      - #           985      - #           990            
  - - Asp Ser Gln His Lys Gly Leu Lys Gly Gly Ar - #g Met Ser Leu Val Phe 
         995          - #      1000           - #     1005                
  - - Gly Cys Arg His Pro Glu Glu Asp His Leu Ty - #r Gln Glu Glu Met Gln 
    1010              - #  1015               - # 1020                    
  - - Glu Met Val Arg Lys Arg Val Leu Phe Gln Va - #l His Thr Gly Tyr Ser 
 1025               1030 - #               1035  - #              1040    
  - - Arg Leu Pro Gly Lys Pro Lys Val Tyr Val Gl - #n Asp Ile Val Gln Lys 
                1045  - #              1050   - #             1055        
  - - Gln Leu Ala Asn Glu Val Leu Ser Val Leu Hi - #s Gly Glu Gln Gly His 
            1060      - #          1065       - #         1070            
  - - Leu Tyr Ile Cys Gly Asp Val Arg Met Ala Ar - #g Asp Val Ala Thr Thr 
        1075          - #      1080           - #     1085                
  - - Leu Lys Lys Leu Val Ala Thr Lys Leu Asn Le - #u Ser Glu Glu Gln Val 
    1090              - #  1095               - # 1100                    
  - - Glu Asp Tyr Phe Phe Gln Leu Lys Ser Gln Ly - #s Arg Tyr His Glu Asp 
 1105               1110 - #               1115  - #              1120    
  - - Ile Phe Gly Ala Val Phe Ser Tyr Gly Ala Ly - #s Lys Gly Ser Ala Leu 
                1125  - #              1130   - #             1135        
  - - Glu Glu Pro Lys Ala Thr Arg Leu                                     
            1140                                                          
  - -  - - <210> SEQ ID NO 3                                              
 <211> LENGTH: 4780                                                       
 <212> TYPE: DNA                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 3                                                   
  - - gagcggacgg gctcatgatg cctcagatct gatccgcatc taacaggctg gc -         
#aatgaaga     60                                                          
   - - tacccagaga atagttcaca tctatcatgc gtcacttcta gacacagcca tc -        
#agacgcat    120                                                          
   - - ctcctcccct ttctgcctga ccttaggaca cgtcccaccg cctctcttga cg -        
#tctgcctg    180                                                          
   - - gtcaaccatc acttccttag agaataagga gagaggcgga tgcaggaaat ca -        
#tgccaccg    240                                                          
   - - acgggccacc agccatgagt gggtgacgct gagctgacgt caaagacaga ga -        
#gggctgaa    300                                                          
   - - gccttgtcag cacctgtcac cccggctcct gctctccgtg tagcctgaag cc -        
#tggatcct    360                                                          
   - - cctggtgaaa tcatcttggc ctgatagcat tgtgaggtct tcagacagga cc -        
#cctcggaa    420                                                          
   - - gctagttacc atggaggatc acatgttcgg tgttcagcaa atccagccca at -        
#gtcatttc    480                                                          
   - - tgttcgtctc ttcaagcgca aagttggggg cctgggattt ctggtgaagg ag -        
#cgggtcag    540                                                          
   - - taagccgccc gtgatcatct ctgacctgat tcgtgggggc gccgcagagc ag -        
#agtggcct    600                                                          
   - - catccaggcc ggagacatca ttcttgcggt caacggccgg cccttggtgg ac -        
#ctgagcta    660                                                          
   - - tgacagcgcc ctggaggtac tcagaggcat tgcctctgag acccacgtgg tc -        
#ctcattct    720                                                          
   - - gaggggccct gaaggtttca ccacgcacct ggagaccacc tttacaggtg at -        
#gggacccc    780                                                          
   - - caagaccatc cgggtgacac agcccctggg tccccccacc aaagccgtgg at -        
#ctgtccca    840                                                          
   - - ccagccaccg gccggcaaag aacagcccct ggcagtggat ggggcctcgg gt -        
#cccgggaa    900                                                          
   - - tgggcctcag catgcctacg atgatgggca ggaggctggc tcactccccc at -        
#gccaacgg    960                                                          
   - - ctggccccag gcccccaggc aggaccccgc gaagaaagca accagagtca gc -        
#ctccaagg   1020                                                          
   - - cagaggggag aacaatgaac tgctcaagga gatagagcct gtgctgagcc tt -        
#ctcaccag   1080                                                          
   - - tgggagcaga ggggtcaagg gaggggcacc tgccaaggca gagatgaaag at -        
#atgggaat   1140                                                          
   - - ccaggtggac agagatttgg acggcaagtc acacaaacct ctgcccctcg gc -        
#gtggagaa   1200                                                          
   - - cgaccgagtc ttcaatgacc tatgggggaa gggcaatgtg cctgtcgtcc tc -        
#aacaaccc   1260                                                          
   - - atattcagag aaggagcagc cccccacctc aggaaaacag tcccccacaa ag -        
#aatggcag   1320                                                          
   - - cccctccaag tgtccacgct tcctcaaggt caagaactgg gagactgagg tg -        
#gttctcac   1380                                                          
   - - tgacaccctc caccttaaga gcacattgga aacgggatgc actgagtaca tc -        
#tgcatggg   1440                                                          
   - - ctccatcatg catccttctc agcatgcaag gaggcctgaa gacgtccgca ca -        
#aaaggaca   1500                                                          
   - - gctcttccct ctcgccaaag agtttattga tcaatactat tcatcaatta aa -        
#agatttgg   1560                                                          
   - - ctccaaagcc cacatggaaa ggctggaaga ggtgaacaaa gagatcgaca cc -        
#actagcac   1620                                                          
   - - ttaccagctc aaggacacag agctcatcta tggggccaag cacgcctggc gg -        
#aatgcctc   1680                                                          
   - - gcgctgtgtg ggcaggatcc agtggtccaa gctgcaggta ttcgatgccc gt -        
#gactgcac   1740                                                          
   - - cacggcccac gggatgttca actacatctg taaccatgtc aagtatgcca cc -        
#aacaaagg   1800                                                          
   - - gaacctcagg tctgccatca ccatattccc ccagaggaca gacggcaagc ac -        
#gacttccg   1860                                                          
   - - agtctggaac tcccagctca tccgctacgc tggctacaag caccgtgacg gc -        
#tccaccct   1920                                                          
   - - gggggaccca gccaatgtgc agttcacaga gatatgcata cagcagggct gg -        
#aaaccgcc   1980                                                          
   - - tagaggccgc ttcgatgtcc tgccgctcct gcttcaggcc aacggcaatg ac -        
#cctgagct   2040                                                          
   - - cttccagatt cctccagagc tggtgttgga acttcccatc aggcacccca ag -        
#tttgagtg   2100                                                          
   - - gttcaaggac ctggcgctga agtggtacgg cctccccgcc gtgtccaaca tg -        
#ctcctaga   2160                                                          
   - - gattggcggc ctggagttca gcgcctgtcc cttcagtggc tggtacatgg gc -        
#acagagat   2220                                                          
   - - tggtgtccgc gactactgtg acaactcccg ctacaatatc ctggaggaag tg -        
#gccaagaa   2280                                                          
   - - gatgaactta gacatgagga agacgtcctc cctgtggaag gaccaggcgc tg -        
#gtggagat   2340                                                          
   - - caatatcgcg gttctctata gcttccagag tgacaaagtg accattgttg ac -        
#catcactc   2400                                                          
   - - cgccaccgag tccttcatta agcacatgga gaatgagtac cgctgccggg gg -        
#ggctgccc   2460                                                          
   - - tgccgactgg gtgtggatcg tgccccccat gtccggaagc atcacccctg tg -        
#ttccacca   2520                                                          
   - - ggagatgctc aactaccggc tcaccccctc cttcgaatac cagcctgatc cc -        
#tggaacac   2580                                                          
   - - gcatgtctgg aaaggcacca acgggacccc cacaaagcgg cgagccatcg gc -        
#ttcaagaa   2640                                                          
   - - gctagcagaa gctgtcaagt tctcggccaa gctgatgggg caggctatgg cc -        
#aagagggt   2700                                                          
   - - gaaagcgacc atcctctatg ccacagagac aggcaaatcg caagcttatg cc -        
#aagacctt   2760                                                          
   - - gtgtgagatc ttcaaacacg cctttgatgc caaggtgatg tccatggaag aa -        
#tatgacat   2820                                                          
   - - tgtgcacctg gaacatgaaa ctctggtcct tgtggtcacc agcacctttg gc -        
#aatggaga   2880                                                          
   - - tccccctgag aatggggaga aattcggctg tgctttgatg gaaatgaggc ac -        
#cccaactc   2940                                                          
   - - tgtgcaggaa gaaaggaaga gctacaaggt ccgattcaac agcgtctcct cc -        
#tactctga   3000                                                          
   - - ctcccaaaaa tcatcaggcg atgggcccga cctcagagac aactttgaga gt -        
#gctggacc   3060                                                          
   - - cctggccaat gtgaggttct cagtttttgg cctcggctca cgagcatacc ct -        
#cacttttg   3120                                                          
   - - cgccttcgga cacgctgtgg acaccctcct ggaagaactg ggaggggaga gg -        
#atcctgaa   3180                                                          
   - - gatgagggaa ggggatgagc tctgtgggca ggaagaggct ttcaggacct gg -        
#gccaagaa   3240                                                          
   - - ggtcttcaag gcagcctgtg atgtcttctg tgtgggagat gatgtcaaca tt -        
#gaaaaggc   3300                                                          
   - - caacaattcc ctcatcagca atgatcgcag ctggaagaga aacaagttcc gc -        
#ctcacctt   3360                                                          
   - - tgtggccgaa gctccagaac tcacacaagg tctatccaat gtccacaaaa ag -        
#cgagtctc   3420                                                          
   - - agctgcccgg ctccttagcc gtcaaaacct ccagagccct aaatccagtc gg -        
#tcaactat   3480                                                          
   - - cttcgtgcgt ctccacacca acgggagcca ggagctgcag taccagcctg gg -        
#gaccacct   3540                                                          
   - - gggtgtcttc cctggcaacc acgaggacct cgtgaatgcc ctgatcgagc gg -        
#ctggagga   3600                                                          
   - - cgcgccgcct gtcaaccaga tggtgaaagt ggaactgctg gaggagcgga ac -        
#acggcttt   3660                                                          
   - - aggtgtcatc agtaactgga cagacgagct ccgcctcccg ccctgcacca tc -        
#ttccaggc   3720                                                          
   - - cttcaagtac tacctggaca tcaccacgcc accaacgcct ctgcagctgc ag -        
#cagtttgc   3780                                                          
   - - ctccctagct accagcgaga aggagaagca gcgtctgctg gtcctcagca ag -        
#ggtttgca   3840                                                          
   - - ggagtacgag gaatggaaat ggggcaagaa ccccaccatc gtggaggtgc tg -        
#gaggagtt   3900                                                          
   - - cccatctatc cagatgccgg ccaccctgct cctgacccag ctgtccctgc tg -        
#cagccccg   3960                                                          
   - - ctactattcc atcagctcct ccccagacat gtaccctgat gaagtgcacc tc -        
#actgtggc   4020                                                          
   - - catcgtttcc taccgcactc gagatggaga aggaccaatt caccacggcg ta -        
#tgctcctc   4080                                                          
   - - ctggctcaac cggatacagg ctgacgaact ggtcccctgt ttcgtgagag ga -        
#gcacccag   4140                                                          
   - - cttccacctg ccccggaacc cccaagtccc ctgcatcctc gttggaccag gc -        
#accggcat   4200                                                          
   - - tgcccctttc cgaagcttct ggcaacagcg gcaatttgat atccaacaca aa -        
#ggaatgaa   4260                                                          
   - - cccctgcccc atggtcctgg tcttcgggtg ccggcaatcc aagatagatc at -        
#atctacag   4320                                                          
   - - ggaagagacc ctgcaggcca agaacaaggg ggtcttcaga gagctgtaca cg -        
#gcttactc   4380                                                          
   - - ccgggagcca gacaaaccaa agaagtacgt gcaggacatc ctgcaggagc ag -        
#ctggcgga   4440                                                          
   - - gtctgtgtac cgagccctga aggagcaagg gggccacata tacgtctgtg gg -        
#gacgtcac   4500                                                          
   - - catggctgct gatgtcctca aagccatcca gcgcatcatg acccagcagg gg -        
#aagctctc   4560                                                          
   - - ggcagaggac gccggcgtat tcatcagccg gatgagggat gacaaccgat ac -        
#catgagga   4620                                                          
   - - tatttttgga gtcaccctgc gaacgatcga agtgaccaac cgccttagat ct -        
#gagtccat   4680                                                          
   - - tgccttcatt gaagagagca aaaaagacac cgatgaggtt ttcagctcct aa -        
#ctggaccc   4740                                                          
   - - tcttgcccag ccggctgcaa gtttgtaagc gcgggacaga     - #                
  - #  4780                                                               
  - -  - - <210> SEQ ID NO 4                                              
 <211> LENGTH: 1433                                                       
 <212> TYPE: PRT                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 4                                                   
  - - Met Glu Asp His Met Phe Gly Val Gln Gln Il - #e Gln Pro Asn Val Ile 
   1               5 - #                 10 - #                 15        
  - - Ser Val Arg Leu Phe Lys Arg Lys Val Gly Gl - #y Leu Gly Phe Leu Val 
              20     - #             25     - #             30            
  - - Lys Glu Arg Val Ser Lys Pro Pro Val Ile Il - #e Ser Asp Leu Ile Arg 
          35         - #         40         - #         45                
  - - Gly Gly Ala Ala Glu Gln Ser Gly Leu Ile Gl - #n Ala Gly Asp Ile Ile 
      50             - #     55             - #     60                    
  - - Leu Ala Val Asn Gly Arg Pro Leu Val Asp Le - #u Ser Tyr Asp Ser Ala 
  65                 - # 70                 - # 75                 - # 80 
  - - Leu Glu Val Leu Arg Gly Ile Ala Ser Glu Th - #r His Val Val Leu Ile 
                  85 - #                 90 - #                 95        
  - - Leu Arg Gly Pro Glu Gly Phe Thr Thr His Le - #u Glu Thr Thr Phe Thr 
             100      - #           105      - #           110            
  - - Gly Asp Gly Thr Pro Lys Thr Ile Arg Val Th - #r Gln Pro Leu Gly Pro 
         115          - #       120          - #       125                
  - - Pro Thr Lys Ala Val Asp Leu Ser His Gln Pr - #o Pro Ala Gly Lys Glu 
     130              - #   135              - #   140                    
  - - Gln Pro Leu Ala Val Asp Gly Ala Ser Gly Pr - #o Gly Asn Gly Pro Gln 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - His Ala Tyr Asp Asp Gly Gln Glu Ala Gly Se - #r Leu Pro His Ala    
Asn                                                                       
                  165  - #               170  - #               175       
  - - Gly Trp Pro Gln Ala Pro Arg Gln Asp Pro Al - #a Lys Lys Ala Thr Arg 
             180      - #           185      - #           190            
  - - Val Ser Leu Gln Gly Arg Gly Glu Asn Asn Gl - #u Leu Leu Lys Glu Ile 
         195          - #       200          - #       205                
  - - Glu Pro Val Leu Ser Leu Leu Thr Ser Gly Se - #r Arg Gly Val Lys Gly 
     210              - #   215              - #   220                    
  - - Gly Ala Pro Ala Lys Ala Glu Met Lys Asp Me - #t Gly Ile Gln Val Asp 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Arg Asp Leu Asp Gly Lys Ser His Lys Pro Le - #u Pro Leu Gly Val    
Glu                                                                       
                  245  - #               250  - #               255       
  - - Asn Asp Arg Val Phe Asn Asp Leu Trp Gly Ly - #s Gly Asn Val Pro Val 
             260      - #           265      - #           270            
  - - Val Leu Asn Asn Pro Tyr Ser Glu Lys Glu Gl - #n Pro Pro Thr Ser Gly 
         275          - #       280          - #       285                
  - - Lys Gln Ser Pro Thr Lys Asn Gly Ser Pro Se - #r Lys Cys Pro Arg Phe 
     290              - #   295              - #   300                    
  - - Leu Lys Val Lys Asn Trp Glu Thr Glu Val Va - #l Leu Thr Asp Thr Leu 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - His Leu Lys Ser Thr Leu Glu Thr Gly Cys Th - #r Glu Tyr Ile Cys    
Met                                                                       
                  325  - #               330  - #               335       
  - - Gly Ser Ile Met His Pro Ser Gln His Ala Ar - #g Arg Pro Glu Asp Val 
             340      - #           345      - #           350            
  - - Arg Thr Lys Gly Gln Leu Phe Pro Leu Ala Ly - #s Glu Phe Ile Asp Gln 
         355          - #       360          - #       365                
  - - Tyr Tyr Ser Ser Ile Lys Arg Phe Gly Ser Ly - #s Ala His Met Glu Arg 
     370              - #   375              - #   380                    
  - - Leu Glu Glu Val Asn Lys Glu Ile Asp Thr Th - #r Ser Thr Tyr Gln Leu 
 385                 3 - #90                 3 - #95                 4 -  
#00                                                                       
   - - Lys Asp Thr Glu Leu Ile Tyr Gly Ala Lys Hi - #s Ala Trp Arg Asn    
Ala                                                                       
                  405  - #               410  - #               415       
  - - Ser Arg Cys Val Gly Arg Ile Gln Trp Ser Ly - #s Leu Gln Val Phe Asp 
             420      - #           425      - #           430            
  - - Ala Arg Asp Cys Thr Thr Ala His Gly Met Ph - #e Asn Tyr Ile Cys Asn 
         435          - #       440          - #       445                
  - - His Val Lys Tyr Ala Thr Asn Lys Gly Asn Le - #u Arg Ser Ala Ile Thr 
     450              - #   455              - #   460                    
  - - Ile Phe Pro Gln Arg Thr Asp Gly Lys His As - #p Phe Arg Val Trp Asn 
 465                 4 - #70                 4 - #75                 4 -  
#80                                                                       
   - - Ser Gln Leu Ile Arg Tyr Ala Gly Tyr Lys Hi - #s Arg Asp Gly Ser    
Thr                                                                       
                  485  - #               490  - #               495       
  - - Leu Gly Asp Pro Ala Asn Val Gln Phe Thr Gl - #u Ile Cys Ile Gln Gln 
             500      - #           505      - #           510            
  - - Gly Trp Lys Pro Pro Arg Gly Arg Phe Asp Va - #l Leu Pro Leu Leu Leu 
         515          - #       520          - #       525                
  - - Gln Ala Asn Gly Asn Asp Pro Glu Leu Phe Gl - #n Ile Pro Pro Glu Leu 
     530              - #   535              - #   540                    
  - - Val Leu Glu Leu Pro Ile Arg His Pro Lys Ph - #e Glu Trp Phe Lys Asp 
 545                 5 - #50                 5 - #55                 5 -  
#60                                                                       
   - - Leu Ala Leu Lys Trp Tyr Gly Leu Pro Ala Va - #l Ser Asn Met Leu    
Leu                                                                       
                  565  - #               570  - #               575       
  - - Glu Ile Gly Gly Leu Glu Phe Ser Ala Cys Pr - #o Phe Ser Gly Trp Tyr 
             580      - #           585      - #           590            
  - - Met Gly Thr Glu Ile Gly Val Arg Asp Tyr Cy - #s Asp Asn Ser Arg Tyr 
         595          - #       600          - #       605                
  - - Asn Ile Leu Glu Glu Val Ala Lys Lys Met As - #n Leu Asp Met Arg Lys 
     610              - #   615              - #   620                    
  - - Thr Ser Ser Leu Trp Lys Asp Gln Ala Leu Va - #l Glu Ile Asn Ile Ala 
 625                 6 - #30                 6 - #35                 6 -  
#40                                                                       
   - - Val Leu Tyr Ser Phe Gln Ser Asp Lys Val Th - #r Ile Val Asp His    
His                                                                       
                  645  - #               650  - #               655       
  - - Ser Ala Thr Glu Ser Phe Ile Lys His Met Gl - #u Asn Glu Tyr Arg Cys 
             660      - #           665      - #           670            
  - - Arg Gly Gly Cys Pro Ala Asp Trp Val Trp Il - #e Val Pro Pro Met Ser 
         675          - #       680          - #       685                
  - - Gly Ser Ile Thr Pro Val Phe His Gln Glu Me - #t Leu Asn Tyr Arg Leu 
     690              - #   695              - #   700                    
  - - Thr Pro Ser Phe Glu Tyr Gln Pro Asp Pro Tr - #p Asn Thr His Val Trp 
 705                 7 - #10                 7 - #15                 7 -  
#20                                                                       
   - - Lys Gly Thr Asn Gly Thr Pro Thr Lys Arg Ar - #g Ala Ile Gly Phe    
Lys                                                                       
                  725  - #               730  - #               735       
  - - Lys Leu Ala Glu Ala Val Lys Phe Ser Ala Ly - #s Leu Met Gly Gln Ala 
             740      - #           745      - #           750            
  - - Met Ala Lys Arg Val Lys Ala Thr Ile Leu Ty - #r Ala Thr Glu Thr Gly 
         755          - #       760          - #       765                
  - - Lys Ser Gln Ala Tyr Ala Lys Thr Leu Cys Gl - #u Ile Phe Lys His Ala 
     770              - #   775              - #   780                    
  - - Phe Asp Ala Lys Val Met Ser Met Glu Glu Ty - #r Asp Ile Val His Leu 
 785                 7 - #90                 7 - #95                 8 -  
#00                                                                       
   - - Glu His Glu Thr Leu Val Leu Val Val Thr Se - #r Thr Phe Gly Asn    
Gly                                                                       
                  805  - #               810  - #               815       
  - - Asp Pro Pro Glu Asn Gly Glu Lys Phe Gly Cy - #s Ala Leu Met Glu Met 
             820      - #           825      - #           830            
  - - Arg His Pro Asn Ser Val Gln Glu Glu Arg Ly - #s Ser Tyr Lys Val Arg 
         835          - #       840          - #       845                
  - - Phe Asn Ser Val Ser Ser Tyr Ser Asp Ser Gl - #n Lys Ser Ser Gly Asp 
     850              - #   855              - #   860                    
  - - Gly Pro Asp Leu Arg Asp Asn Phe Glu Ser Al - #a Gly Pro Leu Ala Asn 
 865                 8 - #70                 8 - #75                 8 -  
#80                                                                       
   - - Val Arg Phe Ser Val Phe Gly Leu Gly Ser Ar - #g Ala Tyr Pro His    
Phe                                                                       
                  885  - #               890  - #               895       
  - - Cys Ala Phe Gly His Ala Val Asp Thr Leu Le - #u Glu Glu Leu Gly Gly 
             900      - #           905      - #           910            
  - - Glu Arg Ile Leu Lys Met Arg Glu Gly Asp Gl - #u Leu Cys Gly Gln Glu 
         915          - #       920          - #       925                
  - - Glu Ala Phe Arg Thr Trp Ala Lys Lys Val Ph - #e Lys Ala Ala Cys Asp 
     930              - #   935              - #   940                    
  - - Val Phe Cys Val Gly Asp Asp Val Asn Ile Gl - #u Lys Ala Asn Asn Ser 
 945                 9 - #50                 9 - #55                 9 -  
#60                                                                       
   - - Leu Ile Ser Asn Asp Arg Ser Trp Lys Arg As - #n Lys Phe Arg Leu    
Thr                                                                       
                  965  - #               970  - #               975       
  - - Phe Val Ala Glu Ala Pro Glu Leu Thr Gln Gl - #y Leu Ser Asn Val His 
             980      - #           985      - #           990            
  - - Lys Lys Arg Val Ser Ala Ala Arg Leu Leu Se - #r Arg Gln Asn Leu Gln 
         995          - #      1000           - #     1005                
  - - Ser Pro Lys Ser Ser Arg Ser Thr Ile Phe Va - #l Arg Leu His Thr Asn 
    1010              - #  1015               - # 1020                    
  - - Gly Ser Gln Glu Leu Gln Tyr Gln Pro Gly As - #p His Leu Gly Val Phe 
 1025               1030 - #               1035  - #              1040    
  - - Pro Gly Asn His Glu Asp Leu Val Asn Ala Le - #u Ile Glu Arg Leu Glu 
                1045  - #              1050   - #             1055        
  - - Asp Ala Pro Pro Val Asn Gln Met Val Lys Va - #l Glu Leu Leu Glu Glu 
            1060      - #          1065       - #         1070            
  - - Arg Asn Thr Ala Leu Gly Val Ile Ser Asn Tr - #p Thr Asp Glu Leu Arg 
        1075          - #      1080           - #     1085                
  - - Leu Pro Pro Cys Thr Ile Phe Gln Ala Phe Ly - #s Tyr Tyr Leu Asp Ile 
    1090              - #  1095               - # 1100                    
  - - Thr Thr Pro Pro Thr Pro Leu Gln Leu Gln Gl - #n Phe Ala Ser Leu Ala 
 1105               1110 - #               1115  - #              1120    
  - - Thr Ser Glu Lys Glu Lys Gln Arg Leu Leu Va - #l Leu Ser Lys Gly Leu 
                1125  - #              1130   - #             1135        
  - - Gln Glu Tyr Glu Glu Trp Lys Trp Gly Lys As - #n Pro Thr Ile Val Glu 
            1140      - #          1145       - #         1150            
  - - Val Leu Glu Glu Phe Pro Ser Ile Gln Met Pr - #o Ala Thr Leu Leu Leu 
        1155          - #      1160           - #     1165                
  - - Thr Gln Leu Ser Leu Leu Gln Pro Arg Tyr Ty - #r Ser Ile Ser Ser Ser 
    1170              - #  1175               - # 1180                    
  - - Pro Asp Met Tyr Pro Asp Glu Val His Leu Th - #r Val Ala Ile Val Ser 
 1185               1190 - #               1195  - #              1200    
  - - Tyr Arg Thr Arg Asp Gly Glu Gly Pro Ile Hi - #s His Gly Val Cys Ser 
                1205  - #              1210   - #             1215        
  - - Ser Trp Leu Asn Arg Ile Gln Ala Asp Glu Le - #u Val Pro Cys Phe Val 
            1220      - #          1225       - #         1230            
  - - Arg Gly Ala Pro Ser Phe His Leu Pro Arg As - #n Pro Gln Val Pro Cys 
        1235          - #      1240           - #     1245                
  - - Ile Leu Val Gly Pro Gly Thr Gly Ile Ala Pr - #o Phe Arg Ser Phe Trp 
    1250              - #  1255               - # 1260                    
  - - Gln Gln Arg Gln Phe Asp Ile Gln His Lys Gl - #y Met Asn Pro Cys Pro 
 1265               1270 - #               1275  - #              1280    
  - - Met Val Leu Val Phe Gly Cys Arg Gln Ser Ly - #s Ile Asp His Ile Tyr 
                1285  - #              1290   - #             1295        
  - - Arg Glu Glu Thr Leu Gln Ala Lys Asn Lys Gl - #y Val Phe Arg Glu Leu 
            1300      - #          1305       - #         1310            
  - - Tyr Thr Ala Tyr Ser Arg Glu Pro Asp Lys Pr - #o Lys Lys Tyr Val Gln 
        1315          - #      1320           - #     1325                
  - - Asp Ile Leu Gln Glu Gln Leu Ala Glu Ser Va - #l Tyr Arg Ala Leu Lys 
    1330              - #  1335               - # 1340                    
  - - Glu Gln Gly Gly His Ile Tyr Val Cys Gly As - #p Val Thr Met Ala Ala 
 1345               1350 - #               1355  - #              1360    
  - - Asp Val Leu Lys Ala Ile Gln Arg Ile Met Th - #r Gln Gln Gly Lys Leu 
                1365  - #              1370   - #             1375        
  - - Ser Ala Glu Asp Ala Gly Val Phe Ile Ser Ar - #g Met Arg Asp Asp Asn 
            1380      - #          1385       - #         1390            
  - - Arg Tyr His Glu Asp Ile Phe Gly Val Thr Le - #u Arg Thr Ile Glu Val 
        1395          - #      1400           - #     1405                
  - - Thr Asn Arg Leu Arg Ser Glu Ser Ile Ala Ph - #e Ile Glu Glu Ser Lys 
    1410              - #  1415               - # 1420                    
  - - Lys Asp Thr Asp Glu Val Phe Ser Ser                                 
 1425               1430                                                  
  - -  - - <210> SEQ ID NO 5                                              
 <211> LENGTH: 4097                                                       
 <212> TYPE: DNA                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 5                                                   
  - - gaattcggca cgaggagcca cagagcagac ggaggccgcc cgtccggccc ca -         
#gcgacatg     60                                                          
   - - ggcaacttga agagtgtggg ccaggagccc gggcccccct gcggcctggg gc -        
#tggggctg    120                                                          
   - - ggcctcgggc tatgcggcaa gcagggccca gcctccccgg cacctgagcc ca -        
#gccgggcc    180                                                          
   - - cccgcacccg ccaccccgca cgcgccagac cacagcccag ctcccaacag cc -        
#ccacgctg    240                                                          
   - - acccggcctc cggaggggcc caagttccct cgcgtgaaga actgggagct gg -        
#ggagcatc    300                                                          
   - - acctacgaca ctctgtgcgc gcagtcccaa caggacgggc cctgcactcc ca -        
#ggcgctgc    360                                                          
   - - ctgggctccc tggtgttgcc ccggaaactg cagacccggc cctccccggg ac -        
#ctccaccc    420                                                          
   - - gctgagcagc tgctgagcca ggccagggac ttcatcaacc agtactacag ct -        
#ccatcaag    480                                                          
   - - aggagcggct cccaggctca cgaggagcgg cttcaggagg tggaggccga gg -        
#tggcatcc    540                                                          
   - - acgggcacca tccacctccg agagagtgag ctggtgttcg gggccaagca gg -        
#cctggcgc    600                                                          
   - - aatgcacccc gctgcgtggg ccgcatccag tgggggaagc tgcaggtgtt tg -        
#atgcccgg    660                                                          
   - - gactgcagct cagcacagga gatgttcacc tacatctgca accacatcaa gt -        
#acgccacc    720                                                          
   - - aaccgcggca accttcgctc ggccatcaca gtgttcccgc agcgcgcccc gg -        
#gccgcgga    780                                                          
   - - gacttccgga tctggaacag ccagctggtg cgctacgcag gctacagaca gc -        
#aggatggc    840                                                          
   - - tctgtgcgtg gggacccagc caacgtggag atcacggagc tctgcatcca gc -        
#acggctgg    900                                                          
   - - acccccggaa acggccgctt cgacgtgctg cccctgctgc tccaggcccc ag -        
#acgaggct    960                                                          
   - - ccagagctct ttgttctgcc ccccgagctg gtccttgaag tgcccctagg ag -        
#caccccac   1020                                                          
   - - actggagtgg ttcgcggccc tgggctgcga tggtatgccc tcccggccgt gt -        
#ccaacatg   1080                                                          
   - - ctgctggaaa tcgggggtct ggagttctcc gcggccccct tcagcggctg gt -        
#acatgagc   1140                                                          
   - - acggagattg gcacgcggaa cctgtgtgac cctcaccgct acaatatcct gg -        
#aggatgtg   1200                                                          
   - - gccgtctgca tggacctcga cacgcggacc acctcgtccc tgtggaagga ca -        
#aggcggcc   1260                                                          
   - - gtggagatca acctggctgt gctgcacagc tttcagctcg ccaaggtgac ca -        
#tcgtggac   1320                                                          
   - - caccacgccg ccacggtgtc cttcatgaag cacctggaca acgagcagaa gg -        
#ccaggggg   1380                                                          
   - - ggctgccccg ccgactgggc ctggatcgtg ccccccatct acggcagcct ac -        
#cgcccgtc   1440                                                          
   - - ttccaccagg agatggtcaa ctacatcctg tcccctgcct tccgctacca gc -        
#cagacccc   1500                                                          
   - - tggaaaggga gcgcgaccaa gggcgccggc atcaccagga agaagacctt ta -        
#aggaagtg   1560                                                          
   - - gccaacgcgg tgaagatctc tgcctcactc atgggcaccc tgatggccaa gc -        
#gagtgaaa   1620                                                          
   - - gcaaccatcc tgtacgcctc tgagaccggc cgggcccaga gctacgctca gc -        
#agctgggg   1680                                                          
   - - aggctcttcc ggaaggcctt cgatccccgg gtcctgtgca tggatgagta tg -        
#acgtggtg   1740                                                          
   - - tccctggagc acgaggcact ggtgctggtg gtgaccagca cctttgggaa tg -        
#gcgatccc   1800                                                          
   - - ccggagaatg gagagagttt tgcagctgcc ctgatggaga tgtcggggcc ct -        
#acaacagc   1860                                                          
   - - tccccacggc cggaacagca caagagttac aagatccgct tcaacagcgt ct -        
#cctgctca   1920                                                          
   - - gacccgctgg tgtcctcctg gcggcggaag agaaaggagt ccagcaacac ag -        
#acagcgcg   1980                                                          
   - - ggggccctgg ggaccctcag gttctgtgtg ttcggactgg gctcccgggc gt -        
#acccccac   2040                                                          
   - - ttctgcgcct tcgcgcgagc ggtggacacc cggctggaag agcttggagg gg -        
#agcggctg   2100                                                          
   - - ctgcagctgg gccagggcga tgagctctgc ggccaggaag aggccttccg tg -        
#gttgggca   2160                                                          
   - - aaggcggcat tccaggcctc ctgcgagacg ttctgcgttg gggaggaggc ca -        
#aggctgct   2220                                                          
   - - gcccaggaca tcttcagccc caaacggagc tggaaacgcc agaggtaccg gc -        
#tgagcgcc   2280                                                          
   - - caggccgagg gcctccagct gctgccaggc ctgatccacg tgcacagacg ga -        
#agatgttt   2340                                                          
   - - caggccacag tcctctcggt ggaaaatctg caaagcagca agtccacccg gg -        
#ccaccatc   2400                                                          
   - - ctggtgcgcc tggacactgc aggccaggag gggctgcagt accagccggg gg -        
#accacata   2460                                                          
   - - ggcatctccg cgcccaaccg gccgggcctg gtggaggcgc tgctgagccg cg -        
#tggaggac   2520                                                          
   - - ccgccaccgc ccaccgagtc tgtggctgtg gagcagctgg agaaaggcag cc -        
#caggcggc   2580                                                          
   - - cctcctccca gctgggtgcg ggacccacgg ctgcccccgt gcaccgtgcg cc -        
#aggctctc   2640                                                          
   - - accttcttcc tggacatcac ctccccaccc agcccccggc ttctccgact gc -        
#tcagcacc   2700                                                          
   - - ctggccgaag aacccagcga gcagcaggag cttgagaccc tcagtcagga cc -        
#cccggcgc   2760                                                          
   - - tacgaggagt ggaagttggt ccgctgcccc acgctgctgg aggtgctgga gc -        
#agttcccg   2820                                                          
   - - tccgtggcgc tgcccgcccc gctgctcctc acccagctgc ccctgctgca gc -        
#cccggtac   2880                                                          
   - - tactctgtca gctcggcccc caacgcccac cccggagagg tccacctcac ag -        
#tggccgtg   2940                                                          
   - - ctggcgtaca ggacccaaga tgggctgggc cccctacact acggggtctg ct -        
#ccacatgg   3000                                                          
   - - ctgagccagc tcaagactgg agaccccgtg ccctgcttca tcaggggggc tc -        
#cctccttc   3060                                                          
   - - cggctgccgc ctgaccccta cgtgccctgc atcctcgtgg gccctggcac tg -        
#gcatcgcc   3120                                                          
   - - cccttccggg gattttggca ggagaggctg catgacattg agagcaaagg gc -        
#tgcagccg   3180                                                          
   - - caccccatga ccctggtgtt cggctgccgc tgctcccaac tcgaccatct ct -        
#accgcgac   3240                                                          
   - - gaggtgcagg acgcccagga gcgcggggtg tttggccgcg tcctcaccgc ct -        
#tctcccgg   3300                                                          
   - - gaacctgaca gccccaagac ctacgtacag gacatcctga gaaccgagct gg -        
#ctgccgag   3360                                                          
   - - gtgcaccgcg tgctgtgcct cgagcggggc cacatgtttg tctgcggcga tg -        
#tcactatg   3420                                                          
   - - gcaaccagcg tcctgcagac ggtgcagcgc atcttggcga cagagggcga ca -        
#tggagctg   3480                                                          
   - - gacgaggcgg gcgacgtcat cggcgtgctg cgggatcagc aacgctatca cg -        
#aggacatt   3540                                                          
   - - ttcggcctca cgctgcgcac ccaggaggtg acaagccgta tacgtaccca ga -        
#gcttttcc   3600                                                          
   - - ctgcaggagc ggcatctgcg gggcgcggtg ccctgggcct tcgacccgcc cg -        
#gcccagac   3660                                                          
   - - acccccggcc cctgagaccc ctcttgcttc ccactgcagt tcccggagag ag -        
#gggctgtc   3720                                                          
   - - attccactat ggctctaccg ctgtcctgtt ggcctttacc gggaccggcc ac -        
#ctctccct   3780                                                          
   - - cccctcccaa ggtgacttcc cagagactgt tggattccct gtactatctc at -        
#cctctcat   3840                                                          
   - - ctctaggtct gtttccccac cctaagtcca tctggaagac ccctcccagc ag -        
#cggtattc   3900                                                          
   - - cagagcctac agtcagccct ttggtgttta ggtgaatttt agattcccct cg -        
#cctctctc   3960                                                          
   - - ccgggaagta tcttatcttg aaacctgatc tctaaatcat tcaaatattt at -        
#tattgaag   4020                                                          
   - - atttaccata agagactgga ccagaagtta ggagacctac taagatgcct aa -        
#gccaaggt   4080                                                          
   - - cctccggggc cgaattc             - #                  - #            
      - # 4097                                                            
  - -  - - <210> SEQ ID NO 6                                              
 <211> LENGTH: 1205                                                       
 <212> TYPE: PRT                                                          
 <213> ORGANISM: Cytomegalovirus                                          
  - - <400> SEQUENCE: 6                                                   
  - - Met Gly Asn Leu Lys Ser Val Gly Gln Glu Pr - #o Gly Pro Pro Cys Gly 
   1               5 - #                 10 - #                 15        
  - - Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gl - #y Lys Gln Gly Pro Ala 
              20     - #             25     - #             30            
  - - Ser Pro Ala Pro Glu Pro Ser Arg Ala Pro Al - #a Pro Ala Thr Pro His 
          35         - #         40         - #         45                
  - - Ala Pro Asp His Ser Pro Ala Pro Asn Ser Pr - #o Thr Leu Thr Arg Pro 
      50             - #     55             - #     60                    
  - - Pro Glu Gly Pro Lys Phe Pro Arg Val Lys As - #n Trp Glu Leu Gly Ser 
  65                 - # 70                 - # 75                 - # 80 
  - - Ile Thr Tyr Asp Thr Leu Cys Ala Gln Ser Gl - #n Gln Asp Gly Pro Cys 
                  85 - #                 90 - #                 95        
  - - Thr Pro Arg Arg Cys Leu Gly Ser Leu Val Le - #u Pro Arg Lys Leu Gln 
             100      - #           105      - #           110            
  - - Thr Arg Pro Ser Pro Gly Pro Pro Pro Ala Gl - #u Gln Leu Leu Ser Gln 
         115          - #       120          - #       125                
  - - Ala Arg Asp Phe Ile Asn Gln Tyr Tyr Ser Se - #r Ile Lys Arg Ser Gly 
     130              - #   135              - #   140                    
  - - Ser Gln Ala His Glu Glu Arg Leu Gln Glu Va - #l Glu Ala Glu Val Ala 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - Ser Thr Gly Thr Ile His Leu Arg Glu Ser Gl - #u Leu Val Phe Gly    
Ala                                                                       
                  165  - #               170  - #               175       
  - - Lys Gln Ala Trp Arg Asn Ala Pro Arg Cys Va - #l Gly Arg Ile Gln Trp 
             180      - #           185      - #           190            
  - - Gly Lys Leu Gln Val Phe Asp Ala Arg Asp Cy - #s Ser Ser Ala Gln Glu 
         195          - #       200          - #       205                
  - - Met Phe Thr Tyr Ile Cys Asn His Ile Lys Ty - #r Ala Thr Asn Arg Gly 
     210              - #   215              - #   220                    
  - - Asn Leu Arg Ser Ala Ile Thr Val Phe Pro Gl - #n Arg Ala Pro Gly Arg 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Gly Asp Phe Arg Ile Trp Asn Ser Gln Leu Va - #l Arg Tyr Ala Gly    
Tyr                                                                       
                  245  - #               250  - #               255       
  - - Arg Gln Gln Asp Gly Ser Val Arg Gly Asp Pr - #o Ala Asn Val Glu Ile 
             260      - #           265      - #           270            
  - - Thr Glu Leu Cys Ile Gln His Gly Trp Thr Pr - #o Gly Asn Gly Arg Phe 
         275          - #       280          - #       285                
  - - Asp Val Leu Pro Leu Leu Leu Gln Ala Pro As - #p Glu Ala Pro Glu Leu 
     290              - #   295              - #   300                    
  - - Phe Val Leu Pro Pro Glu Leu Val Leu Glu Va - #l Pro Leu Gly Ala Pro 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - His Thr Gly Val Val Arg Gly Pro Gly Leu Ar - #g Trp Tyr Ala Leu    
Pro                                                                       
                  325  - #               330  - #               335       
  - - Ala Val Ser Asn Met Leu Leu Glu Ile Gly Gl - #y Leu Glu Phe Ser Ala 
             340      - #           345      - #           350            
  - - Ala Pro Phe Ser Gly Trp Tyr Met Ser Thr Gl - #u Ile Gly Thr Arg Asn 
         355          - #       360          - #       365                
  - - Leu Cys Asp Pro His Arg Tyr Asn Ile Leu Gl - #u Asp Val Ala Val Cys 
     370              - #   375              - #   380                    
  - - Met Asp Leu Asp Thr Arg Thr Thr Ser Ser Le - #u Trp Lys Asp Lys Ala 
 385                 3 - #90                 3 - #95                 4 -  
#00                                                                       
   - - Ala Val Glu Ile Asn Leu Ala Val Leu His Se - #r Phe Gln Leu Ala    
Lys                                                                       
                  405  - #               410  - #               415       
  - - Val Thr Ile Val Asp His His Ala Ala Thr Va - #l Ser Phe Met Lys His 
             420      - #           425      - #           430            
  - - Leu Asp Asn Glu Gln Lys Ala Arg Gly Gly Cy - #s Pro Ala Asp Trp Ala 
         435          - #       440          - #       445                
  - - Trp Ile Val Pro Pro Ile Tyr Gly Ser Leu Pr - #o Pro Val Phe His Gln 
     450              - #   455              - #   460                    
  - - Glu Met Val Asn Tyr Ile Leu Ser Pro Ala Ph - #e Arg Tyr Gln Pro Asp 
 465                 4 - #70                 4 - #75                 4 -  
#80                                                                       
   - - Pro Trp Lys Gly Ser Ala Thr Lys Gly Ala Gl - #y Ile Thr Arg Lys    
Lys                                                                       
                  485  - #               490  - #               495       
  - - Thr Phe Lys Glu Val Ala Asn Ala Val Lys Il - #e Ser Ala Ser Leu Met 
             500      - #           505      - #           510            
  - - Gly Thr Leu Met Ala Lys Arg Val Lys Ala Th - #r Ile Leu Tyr Ala Ser 
         515          - #       520          - #       525                
  - - Glu Thr Gly Arg Ala Gln Ser Tyr Ala Gln Gl - #n Leu Gly Arg Leu Phe 
     530              - #   535              - #   540                    
  - - Arg Lys Ala Phe Asp Pro Arg Val Leu Cys Me - #t Asp Glu Tyr Asp Val 
 545                 5 - #50                 5 - #55                 5 -  
#60                                                                       
   - - Val Ser Leu Glu His Glu Ala Leu Val Leu Va - #l Val Thr Ser Thr    
Phe                                                                       
                  565  - #               570  - #               575       
  - - Gly Asn Gly Asp Pro Pro Glu Asn Gly Glu Se - #r Phe Ala Ala Ala Leu 
             580      - #           585      - #           590            
  - - Met Glu Met Ser Gly Pro Tyr Asn Ser Ser Pr - #o Arg Pro Glu Gln His 
         595          - #       600          - #       605                
  - - Lys Ser Tyr Lys Ile Arg Phe Asn Ser Val Se - #r Cys Ser Asp Pro Leu 
     610              - #   615              - #   620                    
  - - Val Ser Ser Trp Arg Arg Lys Arg Lys Glu Se - #r Ser Asn Thr Asp Ser 
 625                 6 - #30                 6 - #35                 6 -  
#40                                                                       
   - - Ala Gly Ala Leu Gly Thr Leu Arg Phe Cys Va - #l Phe Gly Leu Gly    
Ser                                                                       
                  645  - #               650  - #               655       
  - - Arg Ala Tyr Pro His Phe Cys Ala Phe Ala Ar - #g Ala Val Asp Thr Arg 
             660      - #           665      - #           670            
  - - Leu Glu Glu Leu Gly Gly Glu Arg Leu Leu Gl - #n Leu Gly Gln Gly Asp 
         675          - #       680          - #       685                
  - - Glu Leu Cys Gly Gln Glu Glu Ala Phe Arg Gl - #y Trp Ala Lys Ala Ala 
     690              - #   695              - #   700                    
  - - Phe Gln Ala Ser Cys Glu Thr Phe Cys Val Gl - #y Glu Glu Ala Lys Ala 
 705                 7 - #10                 7 - #15                 7 -  
#20                                                                       
   - - Ala Ala Gln Asp Ile Phe Ser Pro Lys Arg Se - #r Trp Lys Arg Gln    
Arg                                                                       
                  725  - #               730  - #               735       
  - - Tyr Arg Leu Ser Ala Gln Ala Glu Gly Leu Gl - #n Leu Leu Pro Gly Leu 
             740      - #           745      - #           750            
  - - Ile His Val His Arg Arg Lys Met Phe Gln Al - #a Thr Val Leu Ser Val 
         755          - #       760          - #       765                
  - - Glu Asn Leu Gln Ser Ser Lys Ser Thr Arg Al - #a Thr Ile Leu Val Arg 
     770              - #   775              - #   780                    
  - - Leu Asp Thr Ala Gly Gln Glu Gly Leu Gln Ty - #r Gln Pro Gly Asp His 
 785                 7 - #90                 7 - #95                 8 -  
#00                                                                       
   - - Ile Gly Ile Ser Ala Pro Asn Arg Pro Gly Le - #u Val Glu Ala Leu    
Leu                                                                       
                  805  - #               810  - #               815       
  - - Ser Arg Val Glu Asp Pro Pro Pro Pro Thr Gl - #u Ser Val Ala Val Glu 
             820      - #           825      - #           830            
  - - Gln Leu Glu Lys Gly Ser Pro Gly Gly Pro Pr - #o Pro Ser Trp Val Arg 
         835          - #       840          - #       845                
  - - Asp Pro Arg Leu Pro Pro Cys Thr Val Arg Gl - #n Ala Leu Thr Phe Phe 
     850              - #   855              - #   860                    
  - - Leu Asp Ile Thr Ser Pro Pro Ser Pro Arg Le - #u Leu Arg Leu Leu Ser 
 865                 8 - #70                 8 - #75                 8 -  
#80                                                                       
   - - Thr Leu Ala Glu Glu Pro Ser Glu Gln Gln Gl - #u Leu Glu Thr Leu    
Ser                                                                       
                  885  - #               890  - #               895       
  - - Gln Asp Pro Arg Arg Tyr Glu Glu Trp Lys Le - #u Val Arg Cys Pro Thr 
             900      - #           905      - #           910            
  - - Leu Leu Glu Val Leu Glu Gln Phe Pro Ser Va - #l Ala Leu Pro Ala Pro 
         915          - #       920          - #       925                
  - - Leu Leu Leu Thr Gln Leu Pro Leu Leu Gln Pr - #o Arg Tyr Tyr Ser Val 
     930              - #   935              - #   940                    
  - - Ser Ser Ala Pro Asn Ala His Pro Gly Glu Va - #l His Leu Thr Val Ala 
 945                 9 - #50                 9 - #55                 9 -  
#60                                                                       
   - - Val Leu Ala Tyr Arg Thr Gln Asp Gly Leu Gl - #y Pro Leu His Tyr    
Gly                                                                       
                  965  - #               970  - #               975       
  - - Val Cys Ser Thr Trp Leu Ser Gln Leu Lys Th - #r Gly Asp Pro Val Pro 
             980      - #           985      - #           990            
  - - Cys Phe Ile Arg Gly Ala Pro Ser Phe Arg Le - #u Pro Pro Asp Pro Tyr 
         995          - #      1000           - #     1005                
  - - Val Pro Cys Ile Leu Val Gly Pro Gly Thr Gl - #y Ile Ala Pro Phe Arg 
    1010              - #  1015               - # 1020                    
  - - Gly Phe Trp Gln Glu Arg Leu His Asp Ile Gl - #u Ser Lys Gly Leu Gln 
 1025               1030 - #               1035  - #              1040    
  - - Pro His Pro Met Thr Leu Val Phe Gly Cys Ar - #g Cys Ser Gln Leu Asp 
                1045  - #              1050   - #             1055        
  - - His Leu Tyr Arg Asp Glu Val Gln Asp Ala Gl - #n Glu Arg Gly Val Phe 
            1060      - #          1065       - #         1070            
  - - Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pr - #o Asp Ser Pro Lys Thr 
        1075          - #      1080           - #     1085                
  - - Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu Al - #a Ala Glu Val His Arg 
    1090              - #  1095               - # 1100                    
  - - Val Leu Cys Leu Glu Arg Gly His Met Phe Va - #l Cys Gly Asp Val Thr 
 1105               1110 - #               1115  - #              1120    
  - - Met Ala Thr Ser Val Leu Gln Thr Val Gln Ar - #g Ile Leu Ala Thr Glu 
                1125  - #              1130   - #             1135        
  - - Gly Asp Met Glu Leu Asp Glu Ala Gly Asp Va - #l Ile Gly Val Leu Arg 
            1140      - #          1145       - #         1150            
  - - Asp Gln Gln Arg Tyr His Glu Asp Ile Phe Gl - #y Leu Thr Leu Arg Thr 
        1155          - #      1160           - #     1165                
  - - Gln Glu Val Thr Ser Arg Ile Arg Thr Gln Se - #r Phe Ser Leu Gln Glu 
    1170              - #  1175               - # 1180                    
  - - Arg His Leu Arg Gly Ala Val Pro Trp Ala Ph - #e Asp Pro Pro Gly Pro 
 1185               1190 - #               1195  - #              1200    
  - - Asp Thr Pro Gly Pro                                                 
                1205                                                      
__________________________________________________________________________

Claims (9)

What is claimed is:
1. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent comprising a DNA sequence that codes for a protein which possesses the biological activity of inducible nitrogen monoxide synthase (iNOS) and eukaryotic regulation elements, wherein said eukaryotic regulation elements result in the expression of said DNA sequence in eukaryotic cells, and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1, wherein the DNA sequence that codes for a protein which possesses the biological activity of inducible nitrogen monoxide synthase (iNOS) is a cDNA sequence.
3. The pharmaceutical composition according to claim 1 or 2, wherein the DNA or cDNA sequence is derived from mammals.
4. The pharmaceutical composition according to claim 3, wherein the DNA or CDNA sequence constitutes a human DNA or cDNA sequence.
5. The pharmaceutical composition according to claim 1, wherein said eukaryotic regulation elements are derived from the Cytomegalovirus (CMV) promoter and/or enhancer of the early gene.
6. The pharmaceutical composition according to claim 1, wherein said eukaryotic regulation elements are derived from an eukaryotic virus.
7. The pharmaceutical composition according to claim 1, wherein said DNA expression vector represents pSCMV-iNOS according to FIG. 4e.
8. The pharmaceutical composition according to claim 1, wherein said eukaryotic regulation elements are derived from an Adenovirus promoter and/or enhancer element.
9. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent comprising the plasmid pSCMV-iNOS according to FIG. 4e which contains a DNA sequence that codes for a protein which possesses the biological activity of inducible nitrogen monoxide synthase (INOS) and eukaryotic regulation elements, wherein said eukaryotic regulations elements result in the expression of said DNA sequence in eukaryotic cells, and a pharmaceutically acceptable carrier.
US09/123,708 1994-03-31 1998-07-28 DNA expression vectors for use in the gene therapeutic treatment of vascular disorders Expired - Fee Related US6146887A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100342428C (en) * 2001-06-12 2007-10-10 希捷科技有限公司 Head suspension assembly for slider tester
CN106462072A (en) * 2014-05-08 2017-02-22 三菱瓦斯化学株式会社 Lithographic film formation material, composition for lithographic film formation, lithographic film, pattern formation method, and purification method

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658565A (en) * 1994-06-24 1997-08-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
DE4411402A1 (en) * 1994-03-31 1995-10-05 Juergen Schrader DNA expression vectors for use in gene therapy treatment of vascular diseases
US6150500A (en) * 1996-07-12 2000-11-21 Salerno; John C. Activators of endothelial nitric oxide synthase
DE19729769A1 (en) * 1997-07-11 1999-01-14 Cardiogene Gentherapeutische S Transfection system, its manufacture and use in somatic gene therapy
DE19845434C1 (en) * 1998-10-02 2000-03-16 Juergen Schrader Tissue binding peptides and related nucleotide sequences useful for tissue specific gene transfer
SK14692001A3 (en) * 1999-04-16 2002-09-10 Yale University Isolated nucleic acid molecule, isolated nos polypeptide, a simulation method, a treatment method, an identification method and a non-human transgenic animal
DE19944262A1 (en) * 1999-09-15 2001-03-29 Cardiogene Gentherapeutische S Pharmaceutical composition in the form of a nucleic acid-lipid complex, its production and use in gene therapy
US8308708B2 (en) 2003-07-15 2012-11-13 Abbott Cardiovascular Systems Inc. Deployment system for myocardial cellular material

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0441119A2 (en) * 1990-01-09 1991-08-14 Richard D. Levere The use of L-Arginine in the treatment of hypertension and other vascular disorders
WO1992007943A1 (en) * 1990-10-31 1992-05-14 Somatix Therapy Corporation Retroviral vectors useful for gene therapy
US5132407A (en) * 1991-09-26 1992-07-21 Cornell Research Foundation, Inc. Purified inducible nitric oxide synthase flavoprotein
EP0546796A1 (en) * 1991-12-12 1993-06-16 Ajinomoto Co., Inc. Treatment of Atherosclerosis
WO1993018156A1 (en) * 1992-03-05 1993-09-16 The General Hospital Corporation Endothelial nitric oxide synthase
US5252479A (en) * 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
WO1994012645A2 (en) * 1992-11-25 1994-06-09 University Of Pittsburgh Of The Commonwealth System Of Higher Education cDNA CLONE FOR HUMAN INDUCIBLE NITRIC OXYDE SYNTHASE AND PROCESS FOR PREPARING SAME
US5328470A (en) * 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
WO1994023038A1 (en) * 1993-03-26 1994-10-13 The Wellcome Foundation Limited Inducible nitric oxide synthase and gene therefor
WO1994024269A1 (en) * 1993-04-16 1994-10-27 Novo Nordisk A/S A dna sequence encoding nitric oxide synthase
WO1994028721A1 (en) * 1993-06-11 1994-12-22 The Board Of Trustees Of The Leland Stanford Junior University Treatment of vascular degenerative diseases by modulation of endogenous nitric oxide production or activity
US5428070A (en) * 1993-06-11 1995-06-27 The Board Of Trustees Of The Leland Stanford Junior University Treatment of vascular degenerative diseases by modulation of endogenous nitric oxide production of activity
WO1995027070A1 (en) * 1994-03-31 1995-10-12 Schrader Juergen Dna expression vectors for use in the treatment of vascular diseases by gene therapy
WO1996000006A1 (en) * 1994-06-24 1996-01-04 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
WO1998002170A1 (en) * 1996-07-17 1998-01-22 The General Hospital Corporation Method of inducing vasodilation and treating pulmonary hypertension using adenoviral-mediated transfer of the nitric oxide synthase gene
US5766909A (en) * 1992-02-04 1998-06-16 Merck & Co., Inc. DNA encoding inducible nitric oxide synthase

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5328470A (en) * 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
EP0441119A2 (en) * 1990-01-09 1991-08-14 Richard D. Levere The use of L-Arginine in the treatment of hypertension and other vascular disorders
WO1992007943A1 (en) * 1990-10-31 1992-05-14 Somatix Therapy Corporation Retroviral vectors useful for gene therapy
US5132407A (en) * 1991-09-26 1992-07-21 Cornell Research Foundation, Inc. Purified inducible nitric oxide synthase flavoprotein
US5252479A (en) * 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
EP0546796A1 (en) * 1991-12-12 1993-06-16 Ajinomoto Co., Inc. Treatment of Atherosclerosis
US5766909A (en) * 1992-02-04 1998-06-16 Merck & Co., Inc. DNA encoding inducible nitric oxide synthase
WO1993018156A1 (en) * 1992-03-05 1993-09-16 The General Hospital Corporation Endothelial nitric oxide synthase
US5468630A (en) * 1992-11-25 1995-11-21 University Of Pittsburg Of The Commonwealth System Of Higher Education cDNA clone for human inducible nitric oxide synthase and process for preparing same
WO1994012645A2 (en) * 1992-11-25 1994-06-09 University Of Pittsburgh Of The Commonwealth System Of Higher Education cDNA CLONE FOR HUMAN INDUCIBLE NITRIC OXYDE SYNTHASE AND PROCESS FOR PREPARING SAME
WO1994023038A1 (en) * 1993-03-26 1994-10-13 The Wellcome Foundation Limited Inducible nitric oxide synthase and gene therefor
WO1994024269A1 (en) * 1993-04-16 1994-10-27 Novo Nordisk A/S A dna sequence encoding nitric oxide synthase
US5428070A (en) * 1993-06-11 1995-06-27 The Board Of Trustees Of The Leland Stanford Junior University Treatment of vascular degenerative diseases by modulation of endogenous nitric oxide production of activity
WO1994028721A1 (en) * 1993-06-11 1994-12-22 The Board Of Trustees Of The Leland Stanford Junior University Treatment of vascular degenerative diseases by modulation of endogenous nitric oxide production or activity
WO1995027070A1 (en) * 1994-03-31 1995-10-12 Schrader Juergen Dna expression vectors for use in the treatment of vascular diseases by gene therapy
WO1996000006A1 (en) * 1994-06-24 1996-01-04 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
US5658565A (en) * 1994-06-24 1997-08-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
WO1998002170A1 (en) * 1996-07-17 1998-01-22 The General Hospital Corporation Method of inducing vasodilation and treating pulmonary hypertension using adenoviral-mediated transfer of the nitric oxide synthase gene

Non-Patent Citations (83)

* Cited by examiner, † Cited by third party
Title
Biochem, Biophys. Res. Commun., vol. 153, No. 3, Jun. 30, 1988, pp. 1251 1256. *
Biochem, Biophys. Res. Commun., vol. 153, No. 3, Jun. 30, 1988, pp. 1251-1256.
Biochem, Biophysc. Res. Communc., vol. 191, No. 1, Feb. 26, 1993, pp. 89 94. *
Biochem, Biophysc. Res. Communc., vol. 191, No. 1, Feb. 26, 1993, pp. 89-94.
Bucala, et al., "Advanced Glycosylation Products Quench Nitric Oxide and Mediate Defective Endothelium-Dependant Vasodilalation in Experimental Diabetes" 1991 J. Clin. Invest. 87: 432-438.
Bucala, et al., Advanced Glycosylation Products Quench Nitric Oxide and Mediate Defective Endothelium Dependant Vasodilalation in Experimental Diabetes 1991 J. Clin. Invest. 87: 432 438. *
Charles et al.; "Cloning and Expression of a Rat Neuronal Nitric Oxide Synthase Coding Sequence in a Baculovirus/Insect Cell System" Biochemical and Biophysical Research Communications, vol. 196, No. 3, 1481-1489 (1993).
Charles et al.; "Cloning, Characterization and Expression of a cDNA Encoding an Induable Nitric Oxide Synthase From the Human Chondrocye" Proc. Nat'l Acad. Sci USA 1993; Dec. 1, 90(23):11419-11423 (1993), Abstract.
Charles et al.; Cloning and Expression of a Rat Neuronal Nitric Oxide Synthase Coding Sequence in a Baculovirus/Insect Cell System Biochemical and Biophysical Research Communications, vol. 196, No. 3, 1481 1489 (1993). *
Charles et al.; Cloning, Characterization and Expression of a cDNA Encoding an Induable Nitric Oxide Synthase From the Human Chondrocye Proc. Nat l Acad. Sci USA 1993; Dec. 1, 90(23):11419 11423 (1993), Abstract. *
Chester et al., "Low Basal and stimulated Release of Nitric Oxide in Atherosclerotic Epicardial Coronary Arteries" 1990, Lancet 336: 897-900.
Chester et al., Low Basal and stimulated Release of Nitric Oxide in Atherosclerotic Epicardial Coronary Arteries 1990, Lancet 336: 897 900. *
Chin et al., "Inactivation of Endothelial Derived Relaxing Factory by Oxidized Lipoproteins" 1992, J. Clin. Invest. 89:10-18.
Chin et al., Inactivation of Endothelial Derived Relaxing Factory by Oxidized Lipoproteins 1992, J. Clin. Invest. 89:10 18. *
Clowes et al. "Kinetics of Cellular Proliferation After Arterial Injury" (1983) 327-333.
Clowes et al. Kinetics of Cellular Proliferation After Arterial Injury (1983) 327 333. *
Draiper et al., "L-arginine-derived Nitric Oxide and the Cell-Mediated Immune Response" 1991 Res. Immunol. 142:553-602.
Draiper et al., L arginine derived Nitric Oxide and the Cell Mediated Immune Response 1991 Res. Immunol. 142:553 602. *
F o rstermann et al. v Eurpoean Heart Journal (1993) 14 (Supp. I), Isoforms of Nitric Oxide Synthase: Functions in the Cardiovascular System . *
Feldman et al. "Site Specificity of Adernovirus--Medicated Gene Transfer by Hydrogelated Balloon" (Abstract) JACC Feb. 1994:1A-484A (p. 235A).
Feldman et al. Site Specificity of Adernovirus Medicated Gene Transfer by Hydrogelated Balloon (Abstract) JACC Feb. 1994:1A 484A (p. 235A). *
Forstermann et al. v Eurpoean Heart Journal (1993) 14 (Supp. I), "Isoforms of Nitric Oxide Synthase: Functions in the Cardiovascular System".
Geller et al., "Molecular Cloning and Expression of Inducible Nitric Oxide Synthase from Human Hepatocytes" 1993 PNAS 90: 3491-3495.
Geller et al., Molecular Cloning and Expression of Inducible Nitric Oxide Synthase from Human Hepatocytes 1993 PNAS 90: 3491 3495. *
Geller et al.; "Should Surgeons Clone Genes" Archives of Surgery; American Medical Association vol. 128 p. 1212-1220 (Nov. 1993).
Geller et al.; Should Surgeons Clone Genes Archives of Surgery; American Medical Association vol. 128 p. 1212 1220 (Nov. 1993). *
Gen. Pharmac, vol. 21, No. 5, 1990, pp. 575 587. *
Gen. Pharmac, vol. 21, No. 5, 1990, pp. 575-587.
Ignarro et al. "Endothelium-Derived Relaxing Factor Produced and Released from Artery and Vein in Nitric Oxide" 1987 PNAS 84: 9265-9269.
Ignarro et al. Endothelium Derived Relaxing Factor Produced and Released from Artery and Vein in Nitric Oxide 1987 PNAS 84: 9265 9269. *
J. Clin, Invest, vol. 90, 1992, pp. 1168 1172. *
J. Clin, Invest, vol. 90, 1992, pp. 1168-1172.
J. Clin. Invest., vol. 90, 1992, pp. 1248 1253. *
J. Clin. Invest., vol. 90, 1992, pp. 1248-1253.
Janssens et al.; "Cloning and Expression of a cDNA Encoding Human Endothelium-Derived Relaxing Factor/Nitric Oxide Synthase" The Journal of Biological Chemistry; vol. 267, No. 21 Issue of Jul. 25 pp. 14319-14522 (1992).
Janssens et al.; Cloning and Expression of a cDNA Encoding Human Endothelium Derived Relaxing Factor/Nitric Oxide Synthase The Journal of Biological Chemistry; vol. 267, No. 21 Issue of Jul. 25 pp. 14319 14522 (1992). *
Lange et al. Southwestern Internal Medicine Conference:Restenosis (Od. 1983) p. 265 275. *
Lange et al. Southwestern Internal Medicine Conference:Restenosis (Od. 1983) p. 265-275.
Lloyd Jones, M.D. et al.; The Vascular Biology of Nitric Oxide and its Role In Atherogenesis Annu. Rev. Med. 1996, 47:365 75. *
Lloyd-Jones, M.D. et al.; "The Vascular Biology of Nitric Oxide and its Role In Atherogenesis" Annu. Rev. Med. 1996, 47:365-75.
Lowenstein et al.; "Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme" Proc. Nat'l Acad. Sci. USA; vol. 89, pp. 6711-6715, Aug. 1992.
Lowenstein et al.; Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme Proc. Nat l Acad. Sci. USA; vol. 89, pp. 6711 6715, Aug. 1992. *
Lyons et al., "Molecular Cloning and Functional Expression of an Inducible Nitric Oxide Synthase from a Murine Macrophage Cell Line" 1992 J. Biol. Chem. 267:6370-6374.
Lyons et al., Molecular Cloning and Functional Expression of an Inducible Nitric Oxide Synthase from a Murine Macrophage Cell Line 1992 J. Biol. Chem. 267:6370 6374. *
Moncada, et al. 1991, "Nitric Oxide: Physiology, Pathophysiology and Pharmacology Pharmaceuticals Reviews 43: 109-142.
Moncada, et al. 1991, Nitric Oxide: Physiology, Pathophysiology and Pharmacology Pharmaceuticals Reviews 43: 109 142. *
Nabel et al. "Recombinant Gene Expression In Vivo within Endothelial Cells of the Arterial Wall" Science 244: 1342-1344 (1989).
Nabel et al. "Site-specific Gene Expression In Vivo by Direct Gene Transfer into the Arterial Wall" 1990, Science 249:1285-1288.
Nabel et al. Recombinant Gene Expression In Vivo within Endothelial Cells of the Arterial Wall Science 244: 1342 1344 (1989). *
Nabel et al. Site specific Gene Expression In Vivo by Direct Gene Transfer into the Arterial Wall 1990, Science 249:1285 1288. *
Nakane et al.; "Cloned Human Brain Nitric Oxide Synthase is Highly Expressed in Skeletal Muscle" FEBS Letters, vol. 316, No. 2, 175-180; Jan. 1993.
Nakane et al.; Cloned Human Brain Nitric Oxide Synthase is Highly Expressed in Skeletal Muscle FEBS Letters, vol. 316, No. 2, 175 180; Jan. 1993. *
Nakanishi et al. Efficient Intorduction of Contents of Liposomes into Cells Using Hr3 (Sendai Virus) (1985) 399 409. *
Nakanishi et al. Efficient Intorduction of Contents of Liposomes into Cells Using Hr3 (Sendai Virus) (1985) 399-409.
Nature, vol. 333, Jun. 16, 1988, pp. 664 666. *
Nature, vol. 333, Jun. 16, 1988, pp. 664-666.
Nunokawa and Tanaka, "Interferon-Gamma Inhibits Proliferation of Rat Vascular Smooth Muscle Cells by Nitric Oxide Generation" 1992, Biochem Biophys. Res. Comm. 188: 409-415.
Nunokawa and Tanaka, Interferon Gamma Inhibits Proliferation of Rat Vascular Smooth Muscle Cells by Nitric Oxide Generation 1992, Biochem Biophys. Res. Comm. 188: 409 415. *
Nunokawa et al., Cloning of Inducible Nitric Ozide Synthase in Rat Vascular Smooth Muscle Cells, BioChemical and Biophysical Research Communications; vol. 191, No. 1, Feb. 26, 1993, pp. 89 94. *
Nunokawa et al., Cloning of Inducible Nitric Ozide Synthase in Rat Vascular Smooth Muscle Cells, BioChemical and Biophysical Research Communications; vol. 191, No. 1, Feb. 26, 1993, pp. 89-94.
Nussler et al. "Stimulation of Nitric Oxide in Human Hepatocytes by Cytokines" 1992 Faseb. J., 6(5):A1834(#5200).
Nussler et al. Stimulation of Nitric Oxide in Human Hepatocytes by Cytokines 1992 Faseb. J., 6(5):A1834( 5200). *
Nussler, et al. "Stimulation of the Nitric Oxide Synthase Pathway in Human Hepatocytes by Cytokines and Endotoxins" 1992, J. Exp. Med. 176: 261-264.
Nussler, et al. Stimulation of the Nitric Oxide Synthase Pathway in Human Hepatocytes by Cytokines and Endotoxins 1992, J. Exp. Med. 176: 261 264. *
Radomski et al., "The Anti-Aggregating Properties of Vascular Endothelium: Interactions Between Prostacyclin and Nitric Oxide" 1987 Br. J. Pharmac. 92:639-646.
Radomski et al., The Anti Aggregating Properties of Vascular Endothelium: Interactions Between Prostacyclin and Nitric Oxide 1987 Br. J. Pharmac. 92:639 646. *
Sessa; "Molecular Cloning and Expression of a cDNA Encoding Endothelial Cell Nitric Oxide Synthase" The Journal of Biological Chemistry; vol. 267, No. 22, Issue of Aug. 5, pp. 15274-15276, 1992.
Sessa; Molecular Cloning and Expression of a cDNA Encoding Endothelial Cell Nitric Oxide Synthase The Journal of Biological Chemistry; vol. 267, No. 22, Issue of Aug. 5, pp. 15274 15276, 1992. *
Steg et al. "Local Delivery of Adenovirus for Percutaneous Arterial Gener Transfer" Abstract 3554; Circulation vol. 88, No. 4(2) Oct. 1993, p. I-660.
Steg et al. Local Delivery of Adenovirus for Percutaneous Arterial Gene Transfer (abstract) Supplement to Circulation, vol. 88, No. 4, Para 2 (Oct. 1993). *
Steg et al. Local Delivery of Adenovirus for Percutaneous Arterial Gener Transfer Abstract 3554; Circulation vol. 88, No. 4(2) Oct. 1993, p. I 660. *
von der Leyen et al., "Gene Therapy Inhibiting Vascular Lesion: In Vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene" pNAS USA 92 pp. 1137-1141 (Feb. 1995).
von der Leyen et al., "In Vivo Gene Transfer to Prevent Neointima Hyperplasia after Vascular Injury: Effect of Overexpression of Constitutive Nitric Oxide Synthase" 1994 Faseb. J. 8:A802 (#4651).
von der Leyen et al., Gene Therapy Inhibiting Vascular Lesion: In Vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene pNAS USA 92 pp. 1137 1141 (Feb. 1995). *
von der Leyen et al., In Vivo Gene Transfer to Prevent Neointima Hyperplasia after Vascular Injury: Effect of Overexpression of Constitutive Nitric Oxide Synthase 1994 Faseb. J. 8:A802 ( 4651). *
Werner Felmayer et al, Tetrahydrobiopterin Dependent Formation of Nitrite and Nitrate in Murine Fibroblasts 1990 J. Exp. Med.172: 1599 1607. *
Werner-Felmayer et al, "Tetrahydrobiopterin-Dependent Formation of Nitrite and Nitrate in Murine Fibroblasts" 1990 J. Exp. Med.172: 1599-1607.
Wilson et al., "Implantation of Vascular Grafts Lines with Genetically Modified Endothelial Cells" 1989, Science 244:1344-1346.
Wilson et al., Implantation of Vascular Grafts Lines with Genetically Modified Endothelial Cells 1989, Science 244:1344 1346. *
Xie, et al. "Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages" 1992 Science 256:225-228.
Xie, et al. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages 1992 Science 256:225 228. *
Zwiebel et al. "High-Level Recombinant Gene Expression in Rabbit Endothelial Cells Transduced by Retroviral Vectors" 1989, Science 243: 220-222.
Zwiebel et al. High Level Recombinant Gene Expression in Rabbit Endothelial Cells Transduced by Retroviral Vectors 1989, Science 243: 220 222. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100342428C (en) * 2001-06-12 2007-10-10 希捷科技有限公司 Head suspension assembly for slider tester
CN106462072A (en) * 2014-05-08 2017-02-22 三菱瓦斯化学株式会社 Lithographic film formation material, composition for lithographic film formation, lithographic film, pattern formation method, and purification method

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